Producing recombinant proteins in Vibrio natriegens

Producing recombinant proteins in Vibrio natriegens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Producing recombinant proteins in Vibrio natriegens Matthew Smith, José Sánchez Hernández, Simon Messing, Nitya Ramakrishnan, and 23 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4178091/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jul, 2024 Read the published version in Microbial Cell Factories → Version 1 posted 9 You are reading this latest preprint version Abstract The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new prokaryotic recombinant protein expression host is due in part to its short doubling time of < 10 minutes but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli . We successfully incorporated V. natriegens as an additional prokaryotic expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens , we successfully produced several proteins that were difficult or impossible to produce in E. coli . We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens . Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli . Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights Protein-specific increased expression, solubility, and/or reduced aggregation compared to E. coli Optimized protocols for lab efficiency and protein expression Improved yield for specific proteins over E. coli Background It has long been established that recombinant protein expression platforms have advantages and deficiencies specific to the system. Accordingly, multiple systems have been developed with advantages and disadvantages and scientists use the system(s) that best meets their needs. For example, the often-cited advantages of Escherichia coli are low cost, speed, and the ease of genetic manipulation [ 1 , 2 ]. Yet, protein production is often limited in E. coli due to issues in the areas of protein expression, solubility, protein folding, and activity, particularly when expressing large and/or eukaryotic proteins [ 3 ]. Nevertheless, E. coli is typically one of the first choices in a recombinant protein production effort for many research, biotechnology, and pharmaceutical laboratories, due to its many advantages. It is often the only prokaryotic option that laboratories consider, whereas there are multiple eukaryotic expression systems to choose from (e.g. yeast, insect/baculovirus, HEK293, and CHO) [ 3 ]. Thus, the development of V. natriegens as a prokaryotic expression system by Weinstock et al . [ 4 ], was an inflection point in recombinant protein expression research and many reports have followed, exploring the system for a variety of applications [ 5 – 9 ]. Until this recent increase in interest, the fast growth rate of V. natriegens [ 10 ] was one of the more notable attributes of this Gram-negative bacterium that was isolated from a salt march [ 11 ]. Reports have attributed this fast-growing phenotype to the large number of rRNA operons, and thus ribosomes [ 4 , 12 , 13 ] or a higher level of substrate uptake [ 14 ]. Regardless, V. natriegens might have some advantages over E. coli in recombinant protein expression. The recent work cites fast growth rate, metabolic diversity, and the emerging development of tools for genetic manipulation as positive factors for the use of V. natriegens [ 15 , 16 ]. Fast growth rate was also what induced our lab to investigate the use of this system for recombinant protein production. Yet, while we have found the fast growth rate to be useful, perhaps more impactful to our work has been our discovery that V. natriegens serves as a complementary expression system, allowing production of a subset of proteins that we were unable to produce in E. coli . The roadblocks to production in E. coli that were overcome by switching to V. natriegens , suggest some fundamental differences in the translational process between the two systems. While adopting V. natriegens as a parallel prokaryotic expression platform to our customized E. coli system [ 17 ], we encountered and overcame several obstacles that we report here. The protocols we developed are easy to implement, do not involve costly reagents or equipment, and expand the capabilities of V. natriegens as a practical addition to the field of prokaryotic recombinant protein expression. Methods Cloning and bacterial strains DNA constructs for the expression of Hs.KRAS4b(1-169), Hs.KRAS4b(2-169), Hs.NRAS(1-169), Hs.RAF1(52–192), and Hs.NRAS(1-169)-ITD in the format of His6-MBP-tev-POI (MBP, maltose-binding protein; tev, tobacco etch virus protease recognition sequence; POI, protein of interest) were created by subcloning Entry Clones into pDest-566 (Addgene #11517) using Gateway LR clonase per the manufacturer’s instructions (Thermo Fisher Scientific). Plasmid R714-X01 (renamed from NCBI Reference Sequence: NM_144547.2; Uniprot Q8K592; ref. 12), encoding FLAG-Mm.AMHR2(18–142)-His6, was received from the legacy Tuohy laboratory at the Cleveland Clinic. Nanobody expression plasmids were provided by Matt Hall (NCATS, Bethesda, MD). See Table 1 for details of the proteins encoded by the plasmids used in this work. V. natriegens was obtained from Synthetic Genomics, Inc. E. coli expression work was performed with strain BL21 DE3 Star™ (Thermo Fisher Scientific (Waltham, MA)), modified to contain a pRare plasmid (Cm R ) expressing tRNAs (argU, argW, ileX, glyT, leuW, proL, metT, thrT, tyrU and thrU). Our strain is an isolate (TT1) from this parental line that is resistant to a bacteriophage discovered in our lab (unpublished results). Chemicals and media Unless noted otherwise, all chemicals were obtained from MilliporeSigma (Burlington, MA). Instant Ocean™ was from Spectrum Brands (Blacksburg, VA). DMSO was from NEB (Ipswich, MA). Brain Heart Infusion (BHI) Dry media was from Thermo Fisher Scientific. BHI broth medium was prepared as per [ 4 ] (BHI + v2 salts) but without the addition of MgCl 2 (referred to here as BHIv2-Mg). LB-15 agar petri plates were Lysogeny Broth (LB-Miller modified to 15 g/L NaCl) with 2.0% (w/v) agar amended as needed with either 5 µg/mL ampicillin (for initial transformation plates) or 50 µg/mL ampicillin (for colony isolation) for plasmid maintenance. All liquid cultures were amended with 50 µg/mL ampicillin. ZYM-20052 medium [ 17 ] was modified to 1.5% (w/v) Instant Ocean™ with no lactose added (referred to here as ZYM-20050-IO), was used for overnight seed growths. TBV2 medium is (per liter) 12 g tryptone, 24 g yeast extract, 15 g NaCl, 0.5% (v/v) glycerol, 2.31 g KH 2 PO 4 , and 16.43 g K 2 HPO 4 ⋅3H 2 O. The potassium salts are prepared separately in ~ 100 mL dH 2 O (per liter of final volume), filter sterilized, and added to the other components which had been autoclaved separately. Preparation of chemically competent cells Chemically competent V. natriegens cells were prepared as described [ 4 ] by Weinstock et al ., with modifications. Specifically, a glycerol stock was used to obtain an isolated colony by a T-streak on an LB-15 agar plate (no antibiotics) and incubated overnight at 30°C, MgCl 2 was omitted from the BHI broth, and the final pool of competent cells in transformation storage buffer (prepared as described [ 4 ]) was amended with filter sterilized glycerol to 10% (v/v). Transformation of chemically competent cells A vial of V. natriegens competent cells was retrieved from storage at − 80°C and allowed to thaw on ice. One hundred ng of plasmid DNA (isolated from E. coli DH10B T1 Phage-Resistant cells, Thermo Fisher Scientific) was added to the tube of competent cells, mixed by flicking the tube gently (~ 10 times), and incubated on ice for 30 min. During incubation, 1 mL BHIv2-Mg was added to a culture tube (14 mL round bottom Falcon™ tube, 352059) and warmed to 30°C. The cells/DNA mixture was heat shocked in a 42°C water bath (without shaking) for 30 s and returned to ice for 1.5 min. The one milliliter of warmed BHIv2-Mg medium was added to the cell/DNA mixture, and the transformation reaction was transferred from the microcentrifuge tube back to culture tube and incubated at 30°C for 1 h with agitation at 250 rpm (1” throw). Three hundred microliters of the transformation were plated on antibiotic selection plates (LB-15 agar plate with 5 µg/mL ampicillin, pre-warmed to 30°C) and incubated at 30°C overnight. The next morning a colony was picked from the 5 µg/mL ampicillin plate and struck out on an LB-15 agar, 50 µg/mL ampicillin plate to obtain isolated colonies by T-streak and incubated at 30°C. After ~ 6–8 h, colonies were visible. Small-scale growth Fifty milliliters of ZYM-20050-IO with 50 µg/mL ampicillin in a baffled, 250 mL flask was inoculated with an ice chip from a V. natriegens glycerol stock. This seed culture was incubated at 30°C for 14–16 hr with shaking at 250 rpm (1” throw). The following morning, the OD 600 of the overnight culture was measured and used to inoculate 30 mL of TBV2 medium + 50 mg/mL ampicillin in a 250 mL baffled flask (Optimum Growth™, Thomson, Oceanside, CA) to a calculated OD 600 of 0.1. The flask was incubated at 30°C, shaking at 250 rpm (1” throw). The culture was grown to an OD 600 of 1.5-2.0 (~ 2hrs) and protein expression induced with 1 mM of IPTG. The culture was grown for another 6–7 hours and cells were harvested using a 50 mL conical tube at 3900 x g for 25 minutes at 4°C. Fifty milliliter E. coli cultures were grown using the Dynamite medium protocol as described previously [ 17 ]. Large-scale growth for protein production To produce two liters of expression culture, a BioFlo 110 (3-liter vessel, New Brunswick) was prepared the day before the planned growth with a dissolved oxygen (DO) probe (with fresh electrolyte) in two liters of TBV2 medium and autoclaved using a liquid 30 min cycle. The DO probe was attached to the head unit and allowed to polarize overnight (per manufacturer’s instructions). Fifty milliliters of ZYM-20050-IO in a 250 mL baffled flask was inoculated with an ice chip from a V. natriegens glycerol stock. This seed culture was incubated overnight at 30°C and shaken at 250 rpm (1” throw). The following morning the TBV2 medium in the 3-liter production vessel was completed by adding 200 mL of filter sterilized potassium salts, antifoam (Antifoam 204, added at 0.5 mL/L from a 50% (v/v with water) stock), and 50 µg/L ampicillin. The air flow was set to 3 L/min and the agitation was set to 481 rpm. The minimum target DO was set to 20%, controlled by agitation primarily (agitation range was set to 481–600 rpm) and then by controlling supplied O 2 (range set to 0-100%). The overnight culture density was measured by OD 600 and used to inoculate the TBV2 medium in the BioFlo 110 to a calculated OD 600 of 0.1. The culture was grown to an OD 600 of 1.5-2 (~ 2 hr), protein expression induced with 1 mM of IPTG, the culture grown for an additional ~ 4 hr, final OD 600 measured, and the culture harvested at 9000 x g for 30 mins at 4°C. When expression constructs included the CRD domain of RAF1, ZnCl 2 was added at 300 µM approximately 1 hr before induction of protein expression. Cell pellets were either lysed immediately or stored at -80°C. Two-liter E. coli cultures were grown using the Dynamite medium protocol as described previously [ 17 ]. 15 N isotopic labeling of RAF1 RBD-CRD (52–192) Fifty milliliters of ZYM-20050-IO in a 250 mL baffled flask was inoculated with an ice chip from a V. natriegens glycerol stock. This seed culture was incubated overnight at 30°C and shaken at 250 rpm (1” throw). The following morning, one liter of ModM9 [ 18 ] medium (amended to 4 g/L glucose, 15 g/L NaCl, and 50 mM 15 NH 4 Cl, cat # NLM-467, Cambridge Isotope Laboratories, Inc., Tewksbury, MA) was prepared in a four-liter baffled shake flask. The OD 600 of the overnight culture was measured and the amount of overnight culture required to achieve a starting OD 600 of 0.1 was aseptically withdrawn, centrifuged (3900 x g for 15 min at room temperature), resuspended with a small amount of the prepared ModM9, added to the remaining ModM9 culture in the four-liter flask, and incubated at 30°C, 250 rpm (1” throw), for a target OD 600 of 0.5. At ~ 1 hr prior to reaching that density, the culture was amended to 300 µM ZnCl 2 . Upon reaching OD 600 = 0.5, the protein expression was induced by the addition of 1 mM IPTG and shifted to 25°C for overnight incubation. Cells were harvested by centrifugation (9000 x g , 30 min at 4°C). The protocols for isotopic labeling in E. coli were described previously [ 19 ]. Deuterium labeling of KRAS Fifty milliliters of ZYM-20050-IO in a 250 mL baffled flask was inoculated with an ice chip from a V. natriegens glycerol stock. This seed culture was incubated overnight at 30°C and shaken at 250 rpm (1” throw). The following morning, the OD 600 of the overnight culture was measured and used to inoculate a 250 mL baffled flask (50 mL of ZYM-20050-IO working volume) with a starting OD 600 of 0.3 and incubated at 30°C, 250 rpm (1” throw), until reaching an OD 600 of 1.5 (~ 1 hr). Fifty mL of ModM9-1.5IO D 2 O (ModM9 adjusted to 1.5% w/v Instant Ocean™ and made with 100% D 2 O) was added to the flask (for 50% D 2 O in 100 mL of culture) and the 100 mL resulting culture transferred to a 500 mL baffled flask and the culture grown until OD 600 = 1.5 (~ 1 hr). One hundred mL of ModM9-1.5IO D 2 O was added to the culture (for 75% D 2 O in 200 mL of culture), transferred to a one-liter baffled flask and the culture grown until OD 600 = 1.5 (~ 1.5 hr). The cells were harvested by centrifugation (3900 x g , 15 min at room temperature) and resuspended in 100% ModM9 + 15 g/L NaCl in 100% D 2 O, and 250 mL aliquots were placed in four, one-liter baffled shake flasks. The cultures were grown to OD 600 = 0.5, protein expression induced with the addition of 1 mM IPTG, and the incubator temperature shifted to 25°C for overnight incubation. Cells were harvested by centrifugation (9000 x g , 30 min at 4°C). The protocols for deuterium labeling in E. coli were described previously [ 20 ]. Cell lysis Cells were lysed using a Microfluidics 110-EH (Microfluidics, Westwood, MA) by resuspending the cells in 10 mL of lysis buffer per 1000 OD 600 of the final culture and processing at 13,000 PSI (10,000 PSI for E. coli ) for two passes. For RAF1 proteins, Benzonase™ Nuclease HC (cat # 71205-3, Millipore Sigma) was added at 250 units/L of culture and RNase (Qiagen) was added at 350 units/L of culture. Clarification of V. natriegens lysates is best achieved using ultra-centrifugation (104,000 x g ) for 30 min. Alternatively, high-speed centrifugation (30,000 x g , as used for E. coli ) for 90 minutes can be used, but this approach can lead to longer downstream processing times during sample filtration and/or concentrating protein pools for application to preparative SEC columns. Protein purification G-Hs.KRAS4b(1-169), GG-Hs.KRAS4b(2-169) and G-Hs.NRAS(1-169) proteins were purified as described [ 21 ] (Protein production section, page S-4, in Supporting Information). Hs.RAF1(52–192) was purified as described previously [ 22 ]. Nanobody purification was described previously [ 23 ]. FLAG-Mm.AMHR2(18–142)-His6 was purified as described (manuscript in preparation). Intrinsic GTPase measurement. The intrinsic rates for KRAS and NRAS were determined by the Phosphate Sensor assay [ 24 ]. A 2-fold, 8-point standard curve was generated using KH 2 PO 4 with a starting concentration of 6 µM, with the last point being assay buffer (50 mM HEPES, pH 7.3; 150 mM KCl, 1.5 mM MgCl 2 , 5 mM DTT). RAS proteins (6 µM final concentration) were aliquoted into a 384-microplate and the reaction was initiated by the addition of the Phosphate Sensor (4.5 µM final concentration). The phosphate standards were included in the plate. The plate was sealed to protect from the light and read from the bottom in the BioTek Synergy Neo2 (Ex 430/5, Em 450/5) and read every 2 minutes and 20 seconds for 8 hours at 37°C. Intact mass Mass spectrometry was done as previously described [ 25 ]. Briefly, samples were diluted to 0.1 mg/ml and 50 µL analyzed via liquid chromatography coupled on-line with mass spectrometry. High-resolution intact protein mass (MS1) spectra were acquired over a 600–2500 m/z window at 120,000 FT resolution (at 400 m/z) with an AGC target value of 3e + 06 and averaging 4 microscans. Spectra were analyzed by MagTran (Amgen Inc.). Results Transformation and growth Our initial attempts to transform V. natriegens resulted in insufficient transformants (frequently, no colonies arose). We traced the major source of the problem to higher ampicillin sensitivity than previously reported [ 4 ] when using our standard expression constructs which harbor the gene encoding ampicillin resistance. Lowering the ampicillin concentration from 50 µg/mL to 5 µg/mL resulted in sufficient colonies (Fig. 1 A). Subsequent growth of isolated transformants on both solid and liquid media were possible at 50 µg/mL ampicillin, suggesting a delay in some aspect of establishing resistance compared to E. coli . Additionally, we found by using our in-house prepared chemically competent cells, we obtained more transformants as the cell density of our competent cells was higher than commercial sources (i.e. not through an increase in transformation efficiency). We also modified the transformation protocol by eliminating the second heat shock step and shortening the grow out time to one hour. Thus, we shortened the protocol to 90 minutes with reduced manipulation (Fig. 1 B). The next step to evaluate V. natriegens as an alternative protein expression host was to develop a set of protocols that matched or exceeded the high cell densities and recombinant protein expression levels we routinely achieve with E. coli using our modifications [ 17 ] of the auto-induction protocols reported by the Studier lab [ 26 ]. In our E. coli system, cell densities of ~ 15–20 OD 600 after overnight induction at 16°C are routinely achieved. However, while V. natriegens did grow in modified versions of our auto-induction media ZYM-20052 (e.g. ZYM-20052 minus lactose and amended with 1.5% w/v NaCl; ZYM-20050-IO which also lacks lactose and is amended with 1.5% w/v Instant Ocean™ as described in the Methods), we encountered obstacles: 1) auto-induction of protein expression was not observed in ZYM20052, 2) sub-culture growths for protein production from overnight seed cultures were inconsistent, and 3) IPTG-induced protein expression was reduced in the presence of Instant Ocean™. The lack of auto-induction may be due to the absence of a lacY gene (LacY for lactose transport) and/or the absence of a lacZ gene (encoding beta-galactosidase to convert the lactose into the inducer allolactose) based on the genome sequence ( https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001456255.1/ ). To address the inconsistent growth of subcultures for protein production, we empirically determined that growing a 50 mL overnight seed culture (see Methods) in ZYM-20050-IO and subculturing the following day into TBV2 medium for protein production, gave consistent results. Subcultures grown in this way routinely lead to consistent and robust recombinant protein induction and allowed cell harvest 24 hours after starting the overnight seed (Fig. 2 ). Conditions that did not provide consistent subculture growths include growing a 3 mL seed culture in a 24-well block, the lack of Instant Ocean™ in the overnight seed, and the presence of Instant Ocean™ in the subsequent protein production medium. The growth status of the culture seems to be implicated, but we have yet to determine the specific parameter(s) involved. Due to the high growth rate and cell densities of our production cultures, we measured DO levels and found that under standard aeration conditions (no supplemental oxygen), cultures were below 20% DO within 2–3 hr (data not shown). We investigated the effect of supplementing oxygen to maintain 20% DO and while we did see an increase in the culture density, the protein yield was not substantially improved for the one protein we investigated. This suggests that the amount of protein per cell was reduced. However, more investigation is required to determine the validity of this preliminary data as well as assessing whether this is a protein-specific effect. We prefer to use TBV2 medium with supplemental oxygen over ZYM-20052 (minus lactose, + 1.5% w/v NaCl) as our standard protein production medium for V. natriegens as it is a less complicated medium. Additionally, the culture can be harvested within ~ 6-6.5 hr from the inoculation of the production vessel from the overnight seed and within 24 hr from inoculation of the overnight seed. Protein expression and purification With a reproducible and simple set of protocols in place for culturing V. natriegens and inducing protein expression, we compared the protein expression results between E. coli and V. natriegens. We routinely screen new constructs in a small-scale purification platform [ 27 ] for protein expression and purification in E. coli and baculovirus-infected insect cells. We added V. natriegens to the prokaryotic portion of this screen as the same plasmids can be used in both hosts. We discovered several cases where V. natriegens was the preferred expression host based on the results of small-scale screening. In Fig. 3 A, no expression of FLAG-Mm.AMHR2(18–142)-His6 was detected in our standard condition of Dynamite 16°C [ 17 ] whereas expression was strong, albeit mostly insoluble, in V. natriegens . This result allowed the development of a refolding protocol for the purification of the protein (manuscript in preparation). In Fig. 3 B, small-scale purification of four nanobodies to SARS CoV-2 spike [ 23 ] indicated that while protein can be purified from both E. coli and V. natriegens , protein purity is higher from V. natriegens . Also, the elution fractions from the E. coli -expression system contained significant levels of the E. coli chaperone DnaK. This protein frequently co-purifies in our small-scale and large-scale purifications when the target protein is a soluble aggregate (data not shown). Three of the four nanobodies purified from V. natriegens larger scale cultures (2–4 liters) at high purity as expected from the small-scale screen and with acceptable yields in the range of 5.3 to 33.6 mg/L (Fig. 4 , Table 1 ). However, nanobody RBD-1-2G, was the least promising of the nanobodies in both systems. Thus, as a direct comparison of the expression systems at larger scale, we expressed and purified RBD-1-2G from both systems (in 2–4 liters for V. natriegens and in 15 liters for E. coli ). As indicated in Table 1 , both E. coli productions were low yield and only one produced high purity protein as precipitation of the nanobody in the second preparation reduced the purity (not shown). In contrast, the purifications of RBD-1-2G from V. natriegens were much more successful in terms of purity (Fig. 4 B) and yield (Table 1 ), with the added benefits of a 2-liter fermentation rather than 15 liters. Another example of improved protein production from the V. natriegens system is depicted in Fig. 3 C, where TEV protease digestion of the fusion protein, His6-MBP-tev-G-Hs.NRAS(1-169)-ITD [ 28 ], was successful with V. natriegens -expressed material and not with material from E. coli . This result, along with the observation of the presence of DnaK in the E. coli small scale purification screen of the nanobodies, suggests that protein folding is in some way improved when certain proteins are expressed in V. natriegens . A more striking example of differential protein folding is observed when comparing the chromatographic separation of the initial IMAC capture step for His6-MBP-tev-GG-Hs.KRAS4b(2-169) [ 20 , 29 , 30 ]. Figure 4 A depicts the pertinent elution fractions (A 280 trace and SDS-PAGE/Coomassie staining analysis) and the subsequent TEV protease treatment of pooled fractions. A large percentage (> 50%) of the E. coli -purified material is found in a later eluting peak. This material is particularly resistant to TEV protease digestion and migrates in the void volume of analytical SEC column (data not shown), suggesting that this fraction is a soluble aggregate. In contrast, the same protein elutes as a single peak during IMAC when purified from V. natriegens . This material is digested by TEV protease (Fig. 4 A) and migrates as a monomer in SEC (data not shown). Given that the yield for this protein is not dramatically different between the two systems (Table 1 ), the inference is that, while V. natriegens produces less total soluble fusion protein than E. coli , the proportion of correctly folded protein is higher. Table 1 Proteins produced in this work. Expressed protein Purified protein Yield (mg/L) E. coli V. natriegens FLAG-Mm.AMHR2(18–142)-His6 FLAG-Mm.AMHR2(18–142)-His6 nd 1 7.0 +/- 3.3 Nanobody RBD-1-1G RBD-1-1G nd 2 7.5 Nanobody RBD-1-1E RBD-1-1E nd 2 36.6 Nanobody RBD-1-3H RBD-1-3H nd 2 5.3 Nanobody RBD-1-2G RBD-1-2G 0.1, 0.004 0.7 +/- 0.5 His6-MBP-tev-G-Hs.NRAS(1-169)-ITD G-NRAS(1-169)-ITD nd 3 11.5 His6-MBP-tev-GG-Hs.KRAS4b(2-169) GG-Hs.KRAS4b(2-169) 29 +/- 7 41 +/- 4 His6-MBP-tev-G-Hs.NRAS(1-169) G-Hs.NRAS(1-169) 6 +/- 2 25 +/- 14 His6-MBP-tev-RAF1(52–192) Hs.RAF1(52–192) 7 +/- 1 51 +/- 22 “ “ Hs.RAF1(52–192) 15 N 0.6 +/- 0.5 1.4 +/- 0.3 His6-MBP-tev-G-Hs.KRAS4b(1-169) G-Hs.KRAS4b(1-169) D 2 O 2.3 3.8 +/- 0.6 1 Due to the lack of expressed protein, no scale up was possible. 2 Due to the poor prognosis at small-scale screen, no scale up was attempted. 3 Due to the TEV digestion failure of the scale-up prep, no protein was purified. A similar observation of reduced aggregated protein was made when purifying the G-domain of NRAS (Hs.NRAS(1-169)) which has 92% sequence identity with the KRAS4b G-domain. However, while two IMAC elution peaks were also observed with NRAS (both hosts), as observed with KRAS from E. coli , the proportion of the V. natriegens -produced fusion protein recalcitrant to TEV-protease was lower, leading to a significant increase in yield (25 +/- 14 mg/L from V. natriegens ; 6.0 +/- 2 mg/L from E. coli , Table 1 ). These observations led us to attempt to express and purify Hs.RAF1(52–192), also known as RAF1 CR1, from V. natriegens . The yield of this protein from E. coli is also low (less than 10 mg/L) and is a critical reagent in our laboratory. Yields from the V. natriegens system are significantly higher (Table 1 ) and a Tm can be measured for the protein, which is not possible with the E. coli produced protein. We also frequently observe that during purification, steps that require concentrating large pools to smaller volumes (e.g. preparing the sample for preparative SEC) take considerably longer (data not shown) for E. coli- expressed Hs.RAF1(52–192). Isotopic labeling A considerable roadblock in our lab is the practical and inexpensive production of isotopically labeled proteins. When purification yields drop below one milligram/liter, the cost of isotopically labeled proteins becomes prohibitive due to the larger fermentation volume (and thus reagent cost). As others have shown recently, V. natriegens can also be used to produce isotopically labelled proteins [ 6 , 15 ]. Thus, we explored producing isotopically labelled Hs.RAF1(52–192) in V. natriegens , hoping to reduce costs due to the higher yield. This was investigated by comparing the yield from 15 N isotopic labeling fermentations between E. coli and V. natriegens (Table 1 ). While modest, the increase from 0.6 mg/L to 1.4 mg/L does merit the consideration of producing these more expensive reagents in the V. natriegens system. Similarly, we investigated the possibility of producing deuterated proteins in V. natriegens . As indicated in Table 1 , experiments with KRAS4b suggest that V. natriegens can be considered as an option. Activity To assess if the activity of the proteins produced from V. natriegens was similar to those made from E. coli , we measured the GTPase activity of KRAS4b and NRAS purified from the two systems by using a phosphate sensor assay. This assay uses the release of phosphate as a surrogate for GTPase activity [ 24 ]. Measured rates were similar in both cases: KRAS4b ( V. natriegens 70 µM/min vs E. coli 83 µM/min); NRAS ( V. natriegens 71 µM/min vs E. coli 73 µM/min). Discussion We present findings and protocols that should help interested laboratories add V. natriegens to their recombinant protein production workflow. Specifically, our work defines a set of seed and production media and protocols that builds on our previously published work in E. coli [ 17 ] (with modifications to Studier’s auto-induction studies), that are key to a reproducible system. Additionally, our observations of sensitivity to levels of ampicillin lower than previously reported and the effect Instant Ocean™ can have on culture growth, reproducibility, and protein expression, are critical factors to bear in mind. We suspect that at least some of these findings (e.g. ampicillin sensitivity) might be due to differences with the approaches and techniques rather than a difference between isolates of V. natriegens , but that remains to be investigated. Once in place, the potential of the V. natriegens system, with its fast growth rate, was apparent. The ability to generate a cell pellet within 24 hours from seed culture inoculation, substantially changes the timelines, and thus throughput, of the laboratory (Fig. 2 ): pushing the bottleneck to equipment preparation rather than fermentation. While supplemental oxygen is necessary to achieve this timeline, the improvements in expression, solubility and yield reported here, can still be achieved with extended (~ four additional hours) expression at 30°C (data not shown). Overall, we found using V. natriegens as an alternative expression host for recombinant protein expression improved several projects in the lab with no increase in cost or effort. In some cases, the new expression system was responsible for the success of a protein production project (i.e. the target protein could not be produced from E. coli , as was the case for AMHR2, Fig. 3 A) and/or allowed a stalled project to proceed by reducing fermentation volume/costs compared with E. coli (as was the case for NRAS-ITD from E. coli , Fig. 3 C). Similarly, increased yield allowed the consideration of isotopically labelled difficult-to-express proteins that would have been prohibitive to produce in E. coli , as was the case to produce 15 N RAF1(52–192) and deuterated KRAS4b (both reported here) and 13 C, 15 N, and deuterated RAF1(52–192) (data not shown). However, the most intriguing aspect of the V. natriegens system, from our point of view, is the apparent improvement in protein folding suggested by our data. The observations reported here span a range of protein families and expression levels, suggesting the possibility of fundamental differences between E. coli and V. natriegens that might be exploited. Whether this is due to increased chaperones, elevated levels of ribosomes, and/or some other factor(s), is not known. These difficult to express proteins, illustrate the potential benefits from adding this easy to use, genetically amenable, and microbiologically diverse V. natriegens system to the toolbox for recombinant protein production. Conclusions V. natriegens can be an important addition to a protein expression laboratory. While not a replacement for E. coli , V. natriegens produces some proteins at higher yield and with less aggregation. Incorporating V. natriegens into existing small- and large-scale E. coli project workflows is relatively simple and requires no additional equipment or reagents. In so doing, we have been able to complete several projects heretofore stalled in the standard E. coli expression system due to poor yield and/or protein aggregation. Given the diversity of proteins represented in these projects from the limited number of protein families we have investigated, is seems likely that V. natriegens will be useful in the production of additional proteins. Abbreviations IMAC immobilized metal affinity chromatography SEC size exclusion chromatography Declarations Ethics approval and consent to participate. Not applicable Consent for publication . Not applicable Availability of data and materials. All data generated or analysed during this study are included in this published article. Competing interests. The authors declare that they have no competing interests Funding. This work has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract 75N91019D00024. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Authors' contributions VW, CG and JM designed and produced clones. MS, JSH, NR, BH, PVL, JC, and TT carried out expression work. MS, JSH, SM, NR, SP, JM, AJ, PF, MS, MP, SW, J-PD, KS, MD, MH, IP and AC carried out purification of recombinant proteins. MS, JSH, NR, CG, TT, SM, DE and WG conceived and supervised this project and designed experiments. MS, JSH, NR, TT, SM and WG analyzed data. SM and WG wrote the manuscript. All authors edited and approved the final version of the manuscript. References Rosano GL, Morales ES, Ceccarelli EA. New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Sci. 2019;28(8):1412–22. Shilling PJ, Daley DO. Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production. Bio Protoc. 2021;11(16):e4133. Schutz A, et al. A concise guide to choosing suitable gene expression systems for recombinant protein production. STAR Protoc. 2023;4(4):102572. Weinstock MT, et al. Vibrio natriegens as a fast-growing host for molecular biology. Nat Methods. 2016;13(10):849–51. Thoma F, Blombach B. Metabolic engineering of Vibrio natriegens. Essays Biochem. 2021;65(2):381–92. Becker W, Wimberger F, Zangger K. Vibrio natriegens: An Alternative Expression System for the High-Yield Production of Isotopically Labeled Proteins. Biochemistry. 2019;58(25):2799–803. Wu F, et al. Metabolic engineering of fast-growing Vibrio natriegens for efficient pyruvate production. Microb Cell Fact. 2023;22(1):172. Pfeifer E et al. Generation of a Prophage-Free Variant of the Fast-Growing Bacterium Vibrio natriegens. Appl Environ Microbiol, 2019. 85(17). Sun Y, et al. Recombinant Protein Expression Chassis Library of Vibrio natriegens by Fine-Tuning the Expression of T7 RNA Polymerase. ACS Synth Biol. 2023;12(2):555–64. Eagon RG. Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. J Bacteriol. 1962;83(4):736–7. Payne WJ, Eagon RG, Williams AK. Some observations on the physiology of Pseudomonas natriegens nov. spec. Antonie Van Leeuwenhoek. 1961;27:121–8. Des Soye BJ, et al. Establishing a High-Yielding Cell-Free Protein Synthesis Platform Derived from Vibrio natriegens. ACS Synth Biol. 2018;7(9):2245–55. Aiyar SE, Gaal T, Gourse RL. rRNA promoter activity in the fast-growing bacterium Vibrio natriegens. J Bacteriol. 2002;184(5):1349–58. Hoffart E et al. High Substrate Uptake Rates Empower Vibrio natriegens as Production Host for Industrial Biotechnology. Appl Environ Microbiol, 2017. 83(22). Mojica N, et al. Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration. Biochemistry. 2024;63(5):587–98. Xu J, Yang S, Yang L. Vibrio natriegens as a host for rapid biotechnology. Trends Biotechnol. 2022;40(4):381–4. Taylor T, Denson JP, Esposito D. Optimizing Expression and Solubility of Proteins in E. coli Using Modified Media and Induction Parameters. Methods Mol Biol. 2017;1586:65–82. Cai M, et al. A simple and cost-effective protocol for high-yield expression of deuterated and selectively isoleucine/leucine/valine methyl protonated proteins in Escherichia coli grown in shaker flasks. J Biomol NMR. 2021;75(2–3):83–7. Travers T, et al. Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain. Sci Rep. 2018;8(1):8461. Chao FA, et al. Insights into the Cross Talk between Effector and Allosteric Lobes of KRAS from Methyl Conformational Dynamics. J Am Chem Soc. 2022;144(9):4196–205. Kopra K, et al. Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring. Anal Chem. 2020;92(7):4971–9. Lakshman B, et al. Quantitative biophysical analysis defines key components modulating recruitment of the GTPase KRAS to the plasma membrane. J Biol Chem. 2019;294(6):2193–207. Fu Y, et al. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike. PLoS ONE. 2022;17(8):e0272364. Brune M, et al. Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry. 1994;33(27):8262–71. Frank PH, et al. Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases. Protein Expr Purif. 2024;218:106446. Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005;41(1):207–34. Gillette WK, et al. Purify First: rapid expression and purification of proteins from XMRV. Protein Expr Purif. 2011;76(2):238–47. Nelson AC, et al. RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling. J Biol Chem. 2020;295(28):9335–48. Bonsor DA, et al. Structure of the SHOC2-MRAS-PP1C complex provides insights into RAF activation and Noonan syndrome. Nat Struct Mol Biol. 2022;29(10):966–77. Chao FA, et al. Reduced dynamic complexity allows structure elucidation of an excited state of KRAS(G13D). Commun Biol. 2023;6(1):594. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Jul, 2024 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 15 Apr, 2024 Reviews received at journal 15 Apr, 2024 Reviews received at journal 30 Mar, 2024 Reviewers agreed at journal 29 Mar, 2024 Reviewers agreed at journal 28 Mar, 2024 Reviewers invited by journal 28 Mar, 2024 Submission checks completed at journal 28 Mar, 2024 Editor assigned by journal 28 Mar, 2024 First submitted to journal 27 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4178091","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285683067,"identity":"ae02a844-da2a-4038-9a97-7b2a2167f017","order_by":0,"name":"Matthew Smith","email":"","orcid":"","institution":"Frederick National Laboratory for Cancer Research","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Smith","suffix":""},{"id":285683070,"identity":"76d6abf5-a881-4518-945d-b7be6da4612a","order_by":1,"name":"José Sánchez Hernández","email":"","orcid":"","institution":"Frederick National Laboratory 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19:45:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4178091/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4178091/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-024-02455-5","type":"published","date":"2024-07-24T16:15:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54035530,"identity":"775915aa-a8de-4b37-af10-ec6752ae9f32","added_by":"auto","created_at":"2024-04-03 16:59:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":387610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eV. natriegens\u003c/em\u003e antibiotic resistance and transformation.\u0026nbsp; A) Photographs of agar plates spread with \u003cem\u003eV. natriegens\u003c/em\u003e.\u0026nbsp; Empty cells ( ‘-‘ plasmid) were plated on plates with zero or 5 ug/mL of ampicillin. \u003cem\u003eV. natriegens\u003c/em\u003e transformation mixes (‘+’ plasmid, Amp\u003csup\u003er\u003c/sup\u003e) were plated on plates with 5 mg/mL or 50 mg/mL ampicillin. A single isolated colony from the 5 mg/mL ampicillin plate was streak for isolation on a 50 mg/mL ampicillin plate (far right plate). B) Schematic of \u003cem\u003eV. natriegens \u003c/em\u003etransformation protocols. Top path [4]. Bottom path, this work.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Gillette240327figures1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4178091/v1/5a7aafe2db9d8341e467f607.jpg"},{"id":54035529,"identity":"4bbf44b9-d71e-4a60-8062-34c2b308bd7f","added_by":"auto","created_at":"2024-04-03 16:59:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":217862,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of workflow between \u003cem\u003eE. coli\u003c/em\u003e and\u003cem\u003e V. natriegens. \u003c/em\u003eArrows indicate approximate time of procedure + incubation with the procedure noted above the arrow. TFN – transformation, streak – T-streak for isolated colonies, seed – overnight culture from isolated colony (or glycerol stock, preferred).\u003c/p\u003e","description":"","filename":"Gillette240327figures2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4178091/v1/25c87032a3af54632aa0d406.jpg"},{"id":54035528,"identity":"4d20288a-ccca-44b3-a4fe-1b14d8d41cf3","added_by":"auto","created_at":"2024-04-03 16:59:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":383423,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eV. natriegens\u003c/em\u003e protein production. A) Small-scale purification analysis of FLAG-Mm.AMHR2(18-142)-His6. Dyn10 – Dynamite protocol [17] at 10°C. Dynamite protocol at 16°C. M- protein standards (kDa), T – total lysate, L – soluble portion of lysate (column load), FT – column flow through. B) Small-scale purification analysis of four nanobodies from \u003cem\u003eV. natriegens\u003c/em\u003e(top) and \u003cem\u003eE. coli\u003c/em\u003e (bottom). C) TEV protease digestion of His6-MBP-tev-NRAS(1-169) ITD IMAC pools.\u003c/p\u003e","description":"","filename":"Gillette240327figures3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4178091/v1/ec1742d9ac5de1396a0c09c2.jpg"},{"id":54035531,"identity":"c5d2811e-7f1d-492d-8336-dda2528547b3","added_by":"auto","created_at":"2024-04-03 16:59:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":308672,"visible":true,"origin":"","legend":"\u003cp\u003eA) Chromatographic separation of His6-MBP-tev-GG-Hs.KRAS4b(2-169) and subsequent TEV protease treatment of pooled fractions. Above chromatograms are SDS-PAGE/Coomassie-stained gel analyses of elution fractions. Below chromatograms are SDS-PAGE/Coomassie-stained gel analyses of TEV protease reactions of peak pools (arrows). B) SDS-PAGE/Coomassie-stained gel analysis of final proteins produced from \u003cem\u003eV. natriegens\u003c/em\u003e. One microgram of protein analyzed.\u003c/p\u003e","description":"","filename":"Gillette240327figures4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4178091/v1/48e3fabae7d66ea3b2b27df1.jpg"},{"id":61596128,"identity":"e869a488-b080-48eb-8450-3e3faf01cf66","added_by":"auto","created_at":"2024-08-01 17:24:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1959082,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4178091/v1/7fb0fd83-398d-4cb8-8b14-c8c1024f8de9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Producing recombinant proteins in Vibrio natriegens","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eProtein-specific increased expression, solubility, and/or reduced aggregation compared to \u003cem\u003eE. coli\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul\u003e\n \u003cli\u003eOptimized protocols for lab efficiency and protein expression \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eImproved yield for specific proteins over \u003cem\u003eE. coli\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Background","content":"\u003cp\u003eIt has long been established that recombinant protein expression platforms have advantages and deficiencies specific to the system. Accordingly, multiple systems have been developed with advantages and disadvantages and scientists use the system(s) that best meets their needs. For example, the often-cited advantages of \u003cem\u003eEscherichia coli\u003c/em\u003e are low cost, speed, and the ease of genetic manipulation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Yet, protein production is often limited in \u003cem\u003eE. coli\u003c/em\u003e due to issues in the areas of protein expression, solubility, protein folding, and activity, particularly when expressing large and/or eukaryotic proteins [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nevertheless, \u003cem\u003eE. coli\u003c/em\u003e is typically one of the first choices in a recombinant protein production effort for many research, biotechnology, and pharmaceutical laboratories, due to its many advantages. It is often the only prokaryotic option that laboratories consider, whereas there are multiple eukaryotic expression systems to choose from (e.g. yeast, insect/baculovirus, HEK293, and CHO) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, the development of \u003cem\u003eV. natriegens\u003c/em\u003e as a prokaryotic expression system by Weinstock \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], was an inflection point in recombinant protein expression research and many reports have followed, exploring the system for a variety of applications [\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Until this recent increase in interest, the fast growth rate of \u003cem\u003eV. natriegens\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] was one of the more notable attributes of this Gram-negative bacterium that was isolated from a salt march [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Reports have attributed this fast-growing phenotype to the large number of rRNA operons, and thus ribosomes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] or a higher level of substrate uptake [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Regardless, \u003cem\u003eV. natriegens\u003c/em\u003e might have some advantages over \u003cem\u003eE. coli\u003c/em\u003e in recombinant protein expression.\u003c/p\u003e \u003cp\u003eThe recent work cites fast growth rate, metabolic diversity, and the emerging development of tools for genetic manipulation as positive factors for the use of \u003cem\u003eV. natriegens\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Fast growth rate was also what induced our lab to investigate the use of this system for recombinant protein production. Yet, while we have found the fast growth rate to be useful, perhaps more impactful to our work has been our discovery that \u003cem\u003eV. natriegens\u003c/em\u003e serves as a complementary expression system, allowing production of a subset of proteins that we were unable to produce in \u003cem\u003eE. coli\u003c/em\u003e. The roadblocks to production in \u003cem\u003eE. coli\u003c/em\u003e that were overcome by switching to \u003cem\u003eV. natriegens\u003c/em\u003e, suggest some fundamental differences in the translational process between the two systems. While adopting \u003cem\u003eV. natriegens\u003c/em\u003e as a parallel prokaryotic expression platform to our customized \u003cem\u003eE. coli\u003c/em\u003e system [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we encountered and overcame several obstacles that we report here. The protocols we developed are easy to implement, do not involve costly reagents or equipment, and expand the capabilities of \u003cem\u003eV. natriegens\u003c/em\u003e as a practical addition to the field of prokaryotic recombinant protein expression.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCloning and bacterial strains\u003c/h2\u003e \u003cp\u003eDNA constructs for the expression of Hs.KRAS4b(1-169), Hs.KRAS4b(2-169), Hs.NRAS(1-169), Hs.RAF1(52\u0026ndash;192), and Hs.NRAS(1-169)-ITD in the format of His6-MBP-tev-POI (MBP, maltose-binding protein; tev, tobacco etch virus protease recognition sequence; POI, protein of interest) were created by subcloning Entry Clones into pDest-566 (Addgene #11517) using Gateway LR clonase per the manufacturer\u0026rsquo;s instructions (Thermo Fisher Scientific). Plasmid R714-X01 (renamed from NCBI Reference Sequence: NM_144547.2; Uniprot Q8K592; ref. 12), encoding FLAG-Mm.AMHR2(18\u0026ndash;142)-His6, was received from the legacy Tuohy laboratory at the Cleveland Clinic. Nanobody expression plasmids were provided by Matt Hall (NCATS, Bethesda, MD). See Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for details of the proteins encoded by the plasmids used in this work.\u003c/p\u003e \u003cp\u003e \u003cem\u003eV. natriegens\u003c/em\u003e was obtained from Synthetic Genomics, Inc. \u003cem\u003eE. coli\u003c/em\u003e expression work was performed with strain BL21 DE3 Star\u0026trade; (Thermo Fisher Scientific (Waltham, MA)), modified to contain a pRare plasmid (Cm\u003csup\u003eR\u003c/sup\u003e) expressing tRNAs (argU, argW, ileX, glyT, leuW, proL, metT, thrT, tyrU and thrU). Our strain is an isolate (TT1) from this parental line that is resistant to a bacteriophage discovered in our lab (unpublished results).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and media\u003c/h2\u003e \u003cp\u003eUnless noted otherwise, all chemicals were obtained from MilliporeSigma (Burlington, MA). Instant Ocean\u0026trade; was from Spectrum Brands (Blacksburg, VA). DMSO was from NEB (Ipswich, MA). Brain Heart Infusion (BHI) Dry media was from Thermo Fisher Scientific. BHI broth medium was prepared as per [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] (BHI\u0026thinsp;+\u0026thinsp;v2 salts) but without the addition of MgCl\u003csub\u003e2\u003c/sub\u003e (referred to here as BHIv2-Mg). LB-15 agar petri plates were Lysogeny Broth (LB-Miller modified to 15 g/L NaCl) with 2.0% (w/v) agar amended as needed with either 5 \u0026micro;g/mL ampicillin (for initial transformation plates) or 50 \u0026micro;g/mL ampicillin (for colony isolation) for plasmid maintenance. All liquid cultures were amended with 50 \u0026micro;g/mL ampicillin. ZYM-20052 medium [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] was modified to 1.5% (w/v) Instant Ocean\u0026trade; with no lactose added (referred to here as ZYM-20050-IO), was used for overnight seed growths. TBV2 medium is (per liter) 12 g tryptone, 24 g yeast extract, 15 g NaCl, 0.5% (v/v) glycerol, 2.31 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and 16.43 g K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026sdot;3H\u003csub\u003e2\u003c/sub\u003eO. The potassium salts are prepared separately in ~\u0026thinsp;100 mL dH\u003csub\u003e2\u003c/sub\u003eO (per liter of final volume), filter sterilized, and added to the other components which had been autoclaved separately.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of chemically competent cells\u003c/h2\u003e \u003cp\u003eChemically competent \u003cem\u003eV. natriegens\u003c/em\u003e cells were prepared as described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] by Weinstock \u003cem\u003eet al\u003c/em\u003e., with modifications. Specifically, a glycerol stock was used to obtain an isolated colony by a T-streak on an LB-15 agar plate (no antibiotics) and incubated overnight at 30\u0026deg;C, MgCl\u003csub\u003e2\u003c/sub\u003e was omitted from the BHI broth, and the final pool of competent cells in transformation storage buffer (prepared as described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]) was amended with filter sterilized glycerol to 10% (v/v).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTransformation of chemically competent cells\u003c/h2\u003e \u003cp\u003eA vial of \u003cem\u003eV. natriegens\u003c/em\u003e competent cells was retrieved from storage at \u0026minus;\u0026thinsp;80\u0026deg;C and allowed to thaw on ice. One hundred ng of plasmid DNA (isolated from \u003cem\u003eE. coli\u003c/em\u003e DH10B T1 Phage-Resistant cells, Thermo Fisher Scientific) was added to the tube of competent cells, mixed by flicking the tube gently (~\u0026thinsp;10 times), and incubated on ice for 30 min. During incubation, 1 mL BHIv2-Mg was added to a culture tube (14 mL round bottom Falcon\u0026trade; tube, 352059) and warmed to 30\u0026deg;C. The cells/DNA mixture was heat shocked in a 42\u0026deg;C water bath (without shaking) for 30 s and returned to ice for 1.5 min. The one milliliter of warmed BHIv2-Mg medium was added to the cell/DNA mixture, and the transformation reaction was transferred from the microcentrifuge tube back to culture tube and incubated at 30\u0026deg;C for 1 h with agitation at 250 rpm (1\u0026rdquo; throw). Three hundred microliters of the transformation were plated on antibiotic selection plates (LB-15 agar plate with 5 \u0026micro;g/mL ampicillin, pre-warmed to 30\u0026deg;C) and incubated at 30\u0026deg;C overnight. The next morning a colony was picked from the 5 \u0026micro;g/mL ampicillin plate and struck out on an LB-15 agar, 50 \u0026micro;g/mL ampicillin plate to obtain isolated colonies by T-streak and incubated at 30\u0026deg;C. After ~\u0026thinsp;6\u0026ndash;8 h, colonies were visible.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSmall-scale growth\u003c/h2\u003e \u003cp\u003eFifty milliliters of ZYM-20050-IO with 50 \u0026micro;g/mL ampicillin in a baffled, 250 mL flask was inoculated with an ice chip from a \u003cem\u003eV. natriegens\u003c/em\u003e glycerol stock. This seed culture was incubated at 30\u0026deg;C for 14\u0026ndash;16 hr with shaking at 250 rpm (1\u0026rdquo; throw). The following morning, the OD\u003csub\u003e600\u003c/sub\u003e of the overnight culture was measured and used to inoculate 30 mL of TBV2 medium\u0026thinsp;+\u0026thinsp;50 mg/mL ampicillin in a 250 mL baffled flask (Optimum Growth\u0026trade;, Thomson, Oceanside, CA) to a calculated OD\u003csub\u003e600\u003c/sub\u003e of 0.1. The flask was incubated at 30\u0026deg;C, shaking at 250 rpm (1\u0026rdquo; throw). The culture was grown to an OD\u003csub\u003e600\u003c/sub\u003e of 1.5-2.0 (~\u0026thinsp;2hrs) and protein expression induced with 1 mM of IPTG. The culture was grown for another 6\u0026ndash;7 hours and cells were harvested using a 50 mL conical tube at 3900 x \u003cem\u003eg\u003c/em\u003e for 25 minutes at 4\u0026deg;C. Fifty milliliter \u003cem\u003eE. coli\u003c/em\u003e cultures were grown using the Dynamite medium protocol as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLarge-scale growth for protein production\u003c/h2\u003e \u003cp\u003eTo produce two liters of expression culture, a BioFlo 110 (3-liter vessel, New Brunswick) was prepared the day before the planned growth with a dissolved oxygen (DO) probe (with fresh electrolyte) in two liters of TBV2 medium and autoclaved using a liquid 30 min cycle. The DO probe was attached to the head unit and allowed to polarize overnight (per manufacturer\u0026rsquo;s instructions). Fifty milliliters of ZYM-20050-IO in a 250 mL baffled flask was inoculated with an ice chip from a \u003cem\u003eV. natriegens\u003c/em\u003e glycerol stock. This seed culture was incubated overnight at 30\u0026deg;C and shaken at 250 rpm (1\u0026rdquo; throw). The following morning the TBV2 medium in the 3-liter production vessel was completed by adding 200 mL of filter sterilized potassium salts, antifoam (Antifoam 204, added at 0.5 mL/L from a 50% (v/v with water) stock), and 50 \u0026micro;g/L ampicillin. The air flow was set to 3 L/min and the agitation was set to 481 rpm. The minimum target DO was set to 20%, controlled by agitation primarily (agitation range was set to 481\u0026ndash;600 rpm) and then by controlling supplied O\u003csub\u003e2\u003c/sub\u003e (range set to 0-100%). The overnight culture density was measured by OD\u003csub\u003e600\u003c/sub\u003e and used to inoculate the TBV2 medium in the BioFlo 110 to a calculated OD\u003csub\u003e600\u003c/sub\u003e of 0.1. The culture was grown to an OD\u003csub\u003e600\u003c/sub\u003e of 1.5-2 (~\u0026thinsp;2 hr), protein expression induced with 1 mM of IPTG, the culture grown for an additional\u0026thinsp;~\u0026thinsp;4 hr, final OD\u003csub\u003e600\u003c/sub\u003e measured, and the culture harvested at 9000 x \u003cem\u003eg\u003c/em\u003e for 30 mins at 4\u0026deg;C. When expression constructs included the CRD domain of RAF1, ZnCl\u003csub\u003e2\u003c/sub\u003e was added at 300 \u0026micro;M approximately 1 hr before induction of protein expression. Cell pellets were either lysed immediately or stored at -80\u0026deg;C. Two-liter \u003cem\u003eE. coli\u003c/em\u003e cultures were grown using the Dynamite medium protocol as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003e15\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eN isotopic labeling of RAF1 RBD-CRD (52\u0026ndash;192)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFifty milliliters of ZYM-20050-IO in a 250 mL baffled flask was inoculated with an ice chip from a \u003cem\u003eV. natriegens\u003c/em\u003e glycerol stock. This seed culture was incubated overnight at 30\u0026deg;C and shaken at 250 rpm (1\u0026rdquo; throw). The following morning, one liter of ModM9 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] medium (amended to 4 g/L glucose, 15 g/L NaCl, and 50 mM \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl, cat # NLM-467, Cambridge Isotope Laboratories, Inc., Tewksbury, MA) was prepared in a four-liter baffled shake flask. The OD\u003csub\u003e600\u003c/sub\u003e of the overnight culture was measured and the amount of overnight culture required to achieve a starting OD\u003csub\u003e600\u003c/sub\u003e of 0.1 was aseptically withdrawn, centrifuged (3900 x \u003cem\u003eg\u003c/em\u003e for 15 min at room temperature), resuspended with a small amount of the prepared ModM9, added to the remaining ModM9 culture in the four-liter flask, and incubated at 30\u0026deg;C, 250 rpm (1\u0026rdquo; throw), for a target OD\u003csub\u003e600\u003c/sub\u003e of 0.5. At ~\u0026thinsp;1 hr prior to reaching that density, the culture was amended to 300 \u0026micro;M ZnCl\u003csub\u003e2\u003c/sub\u003e. Upon reaching OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5, the protein expression was induced by the addition of 1 mM IPTG and shifted to 25\u0026deg;C for overnight incubation. Cells were harvested by centrifugation (9000 x \u003cem\u003eg\u003c/em\u003e, 30 min at 4\u0026deg;C). The protocols for isotopic labeling in \u003cem\u003eE. coli\u003c/em\u003e were described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDeuterium labeling of KRAS\u003c/h2\u003e \u003cp\u003eFifty milliliters of ZYM-20050-IO in a 250 mL baffled flask was inoculated with an ice chip from a \u003cem\u003eV. natriegens\u003c/em\u003e glycerol stock. This seed culture was incubated overnight at 30\u0026deg;C and shaken at 250 rpm (1\u0026rdquo; throw). The following morning, the OD\u003csub\u003e600\u003c/sub\u003e of the overnight culture was measured and used to inoculate a 250 mL baffled flask (50 mL of ZYM-20050-IO working volume) with a starting OD\u003csub\u003e600\u003c/sub\u003e of 0.3 and incubated at 30\u0026deg;C, 250 rpm (1\u0026rdquo; throw), until reaching an OD\u003csub\u003e600\u003c/sub\u003e of 1.5 (~\u0026thinsp;1 hr). Fifty mL of ModM9-1.5IO D\u003csub\u003e2\u003c/sub\u003eO (ModM9 adjusted to 1.5% w/v Instant Ocean\u0026trade; and made with 100% D\u003csub\u003e2\u003c/sub\u003eO) was added to the flask (for 50% D\u003csub\u003e2\u003c/sub\u003eO in 100 mL of culture) and the 100 mL resulting culture transferred to a 500 mL baffled flask and the culture grown until OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.5 (~\u0026thinsp;1 hr). One hundred mL of ModM9-1.5IO D\u003csub\u003e2\u003c/sub\u003eO was added to the culture (for 75% D\u003csub\u003e2\u003c/sub\u003eO in 200 mL of culture), transferred to a one-liter baffled flask and the culture grown until OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.5 (~\u0026thinsp;1.5 hr). The cells were harvested by centrifugation (3900 x \u003cem\u003eg\u003c/em\u003e, 15 min at room temperature) and resuspended in 100% ModM9\u0026thinsp;+\u0026thinsp;15 g/L NaCl in 100% D\u003csub\u003e2\u003c/sub\u003eO, and 250 mL aliquots were placed in four, one-liter baffled shake flasks. The cultures were grown to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5, protein expression induced with the addition of 1 mM IPTG, and the incubator temperature shifted to 25\u0026deg;C for overnight incubation. Cells were harvested by centrifugation (9000 x \u003cem\u003eg\u003c/em\u003e, 30 min at 4\u0026deg;C). The protocols for deuterium labeling in \u003cem\u003eE. coli\u003c/em\u003e were described previously [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell lysis\u003c/h2\u003e \u003cp\u003eCells were lysed using a Microfluidics 110-EH (Microfluidics, Westwood, MA) by resuspending the cells in 10 mL of lysis buffer per 1000 OD\u003csub\u003e600\u003c/sub\u003e of the final culture and processing at 13,000 PSI (10,000 PSI for \u003cem\u003eE. coli\u003c/em\u003e) for two passes. For RAF1 proteins, Benzonase\u0026trade; Nuclease HC (cat # 71205-3, Millipore Sigma) was added at 250 units/L of culture and RNase (Qiagen) was added at 350 units/L of culture. Clarification of \u003cem\u003eV. natriegens\u003c/em\u003e lysates is best achieved using ultra-centrifugation (104,000 x \u003cem\u003eg\u003c/em\u003e) for 30 min. Alternatively, high-speed centrifugation (30,000 x \u003cem\u003eg\u003c/em\u003e, as used for \u003cem\u003eE. coli\u003c/em\u003e) for 90 minutes can be used, but this approach can lead to longer downstream processing times during sample filtration and/or concentrating protein pools for application to preparative SEC columns.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein purification\u003c/h2\u003e \u003cp\u003eG-Hs.KRAS4b(1-169), GG-Hs.KRAS4b(2-169) and G-Hs.NRAS(1-169) proteins were purified as described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] (Protein production section, page S-4, in Supporting Information). Hs.RAF1(52\u0026ndash;192) was purified as described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Nanobody purification was described previously [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. FLAG-Mm.AMHR2(18\u0026ndash;142)-His6 was purified as described (manuscript in preparation).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIntrinsic GTPase measurement.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe intrinsic rates for KRAS and NRAS were determined by the Phosphate Sensor assay [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A 2-fold, 8-point standard curve was generated using KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e with a starting concentration of 6 \u0026micro;M, with the last point being assay buffer (50 mM HEPES, pH 7.3; 150 mM KCl, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5 mM DTT). RAS proteins (6 \u0026micro;M final concentration) were aliquoted into a 384-microplate and the reaction was initiated by the addition of the Phosphate Sensor (4.5 \u0026micro;M final concentration). The phosphate standards were included in the plate. The plate was sealed to protect from the light and read from the bottom in the BioTek Synergy Neo2 (Ex 430/5, Em 450/5) and read every 2 minutes and 20 seconds for 8 hours at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIntact mass\u003c/h2\u003e \u003cp\u003eMass spectrometry was done as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, samples were diluted to 0.1 mg/ml and 50 \u0026micro;L analyzed via liquid chromatography coupled on-line with mass spectrometry. High-resolution intact protein mass (MS1) spectra were acquired over a 600\u0026ndash;2500 m/z window at 120,000 FT resolution (at 400 m/z) with an AGC target value of 3e\u0026thinsp;+\u0026thinsp;06 and averaging 4 microscans. Spectra were analyzed by MagTran (Amgen Inc.).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTransformation and growth\u003c/h2\u003e \u003cp\u003eOur initial attempts to transform \u003cem\u003eV. natriegens\u003c/em\u003e resulted in insufficient transformants (frequently, no colonies arose). We traced the major source of the problem to higher ampicillin sensitivity than previously reported [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] when using our standard expression constructs which harbor the gene encoding ampicillin resistance. Lowering the ampicillin concentration from 50 \u0026micro;g/mL to 5 \u0026micro;g/mL resulted in sufficient colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Subsequent growth of isolated transformants on both solid and liquid media were possible at 50 \u0026micro;g/mL ampicillin, suggesting a delay in some aspect of establishing resistance compared to \u003cem\u003eE. coli\u003c/em\u003e. Additionally, we found by using our in-house prepared chemically competent cells, we obtained more transformants as the cell density of our competent cells was higher than commercial sources (i.e. not through an increase in transformation efficiency). We also modified the transformation protocol by eliminating the second heat shock step and shortening the grow out time to one hour. Thus, we shortened the protocol to 90 minutes with reduced manipulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe next step to evaluate \u003cem\u003eV. natriegens\u003c/em\u003e as an alternative protein expression host was to develop a set of protocols that matched or exceeded the high cell densities and recombinant protein expression levels we routinely achieve with \u003cem\u003eE. coli\u003c/em\u003e using our modifications [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] of the auto-induction protocols reported by the Studier lab [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our \u003cem\u003eE. coli\u003c/em\u003e system, cell densities of ~\u0026thinsp;15\u0026ndash;20 OD\u003csub\u003e600\u003c/sub\u003e after overnight induction at 16\u0026deg;C are routinely achieved. However, while \u003cem\u003eV. natriegens\u003c/em\u003e did grow in modified versions of our auto-induction media ZYM-20052 (e.g. ZYM-20052 minus lactose and amended with 1.5% w/v NaCl; ZYM-20050-IO which also lacks lactose and is amended with 1.5% w/v Instant Ocean\u0026trade; as described in the Methods), we encountered obstacles: 1) auto-induction of protein expression was not observed in ZYM20052, 2) sub-culture growths for protein production from overnight seed cultures were inconsistent, and 3) IPTG-induced protein expression was reduced in the presence of Instant Ocean\u0026trade;. The lack of auto-induction may be due to the absence of a \u003cem\u003elacY\u003c/em\u003e gene (LacY for lactose transport) and/or the absence of a \u003cem\u003elacZ\u003c/em\u003e gene (encoding beta-galactosidase to convert the lactose into the inducer allolactose) based on the genome sequence (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001456255.1/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001456255.1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To address the inconsistent growth of subcultures for protein production, we empirically determined that growing a 50 mL overnight seed culture (see Methods) in ZYM-20050-IO and subculturing the following day into TBV2 medium for protein production, gave consistent results. Subcultures grown in this way routinely lead to consistent and robust recombinant protein induction and allowed cell harvest 24 hours after starting the overnight seed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Conditions that did not provide consistent subculture growths include growing a 3 mL seed culture in a 24-well block, the lack of Instant Ocean\u0026trade; in the overnight seed, and the presence of Instant Ocean\u0026trade; in the subsequent protein production medium. The growth status of the culture seems to be implicated, but we have yet to determine the specific parameter(s) involved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDue to the high growth rate and cell densities of our production cultures, we measured DO levels and found that under standard aeration conditions (no supplemental oxygen), cultures were below 20% DO within 2\u0026ndash;3 hr (data not shown). We investigated the effect of supplementing oxygen to maintain 20% DO and while we did see an increase in the culture density, the protein yield was not substantially improved for the one protein we investigated. This suggests that the amount of protein per cell was reduced. However, more investigation is required to determine the validity of this preliminary data as well as assessing whether this is a protein-specific effect. We prefer to use TBV2 medium with supplemental oxygen over ZYM-20052 (minus lactose, +\u0026thinsp;1.5% w/v NaCl) as our standard protein production medium for \u003cem\u003eV. natriegens\u003c/em\u003e as it is a less complicated medium. Additionally, the culture can be harvested within ~\u0026thinsp;6-6.5 hr from the inoculation of the production vessel from the overnight seed and within 24 hr from inoculation of the overnight seed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cp\u003eWith a reproducible and simple set of protocols in place for culturing \u003cem\u003eV. natriegens\u003c/em\u003e and inducing protein expression, we compared the protein expression results between \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eV. natriegens.\u003c/em\u003e We routinely screen new constructs in a small-scale purification platform [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] for protein expression and purification in \u003cem\u003eE. coli\u003c/em\u003e and baculovirus-infected insect cells. We added \u003cem\u003eV. natriegens\u003c/em\u003e to the prokaryotic portion of this screen as the same plasmids can be used in both hosts. We discovered several cases where \u003cem\u003eV. natriegens\u003c/em\u003e was the preferred expression host based on the results of small-scale screening. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, no expression of FLAG-Mm.AMHR2(18\u0026ndash;142)-His6 was detected in our standard condition of Dynamite 16\u0026deg;C [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] whereas expression was strong, albeit mostly insoluble, in \u003cem\u003eV. natriegens\u003c/em\u003e. This result allowed the development of a refolding protocol for the purification of the protein (manuscript in preparation). In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, small-scale purification of four nanobodies to SARS CoV-2 spike [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] indicated that while protein can be purified from both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eV. natriegens\u003c/em\u003e, protein purity is higher from \u003cem\u003eV. natriegens\u003c/em\u003e. Also, the elution fractions from the \u003cem\u003eE. coli\u003c/em\u003e-expression system contained significant levels of the \u003cem\u003eE. coli\u003c/em\u003e chaperone DnaK. This protein frequently co-purifies in our small-scale and large-scale purifications when the target protein is a soluble aggregate (data not shown). Three of the four nanobodies purified from \u003cem\u003eV. natriegens\u003c/em\u003e larger scale cultures (2\u0026ndash;4 liters) at high purity as expected from the small-scale screen and with acceptable yields in the range of 5.3 to 33.6 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, nanobody RBD-1-2G, was the least promising of the nanobodies in both systems. Thus, as a direct comparison of the expression systems at larger scale, we expressed and purified RBD-1-2G from both systems (in 2\u0026ndash;4 liters for \u003cem\u003eV. natriegens\u003c/em\u003e and in 15 liters for \u003cem\u003eE. coli\u003c/em\u003e). As indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, both \u003cem\u003eE. coli\u003c/em\u003e productions were low yield and only one produced high purity protein as precipitation of the nanobody in the second preparation reduced the purity (not shown). In contrast, the purifications of RBD-1-2G from \u003cem\u003eV. natriegens\u003c/em\u003e were much more successful in terms of purity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and yield (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with the added benefits of a 2-liter fermentation rather than 15 liters.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnother example of improved protein production from the \u003cem\u003eV. natriegens\u003c/em\u003e system is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, where TEV protease digestion of the fusion protein, His6-MBP-tev-G-Hs.NRAS(1-169)-ITD [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], was successful with \u003cem\u003eV. natriegens\u003c/em\u003e-expressed material and not with material from \u003cem\u003eE. coli\u003c/em\u003e. This result, along with the observation of the presence of DnaK in the \u003cem\u003eE. coli\u003c/em\u003e small scale purification screen of the nanobodies, suggests that protein folding is in some way improved when certain proteins are expressed in \u003cem\u003eV. natriegens\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eA more striking example of differential protein folding is observed when comparing the chromatographic separation of the initial IMAC capture step for His6-MBP-tev-GG-Hs.KRAS4b(2-169) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA depicts the pertinent elution fractions (A\u003csub\u003e280\u003c/sub\u003e trace and SDS-PAGE/Coomassie staining analysis) and the subsequent TEV protease treatment of pooled fractions. A large percentage (\u0026gt;\u0026thinsp;50%) of the \u003cem\u003eE. coli\u003c/em\u003e-purified material is found in a later eluting peak. This material is particularly resistant to TEV protease digestion and migrates in the void volume of analytical SEC column (data not shown), suggesting that this fraction is a soluble aggregate. In contrast, the same protein elutes as a single peak during IMAC when purified from \u003cem\u003eV. natriegens\u003c/em\u003e. This material is digested by TEV protease (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and migrates as a monomer in SEC (data not shown). Given that the yield for this protein is not dramatically different between the two systems (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the inference is that, while \u003cem\u003eV. natriegens\u003c/em\u003e produces less total soluble fusion protein than \u003cem\u003eE. coli\u003c/em\u003e, the proportion of correctly folded protein is higher.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProteins produced in this work.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExpressed protein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePurified protein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eYield (mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV. natriegens\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFLAG-Mm.AMHR2(18\u0026ndash;142)-His6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFLAG-Mm.AMHR2(18\u0026ndash;142)-His6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.0 +/- 3.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNanobody RBD-1-1G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRBD-1-1G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNanobody RBD-1-1E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRBD-1-1E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNanobody RBD-1-3H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRBD-1-3H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNanobody RBD-1-2G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRBD-1-2G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1, 0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7 +/- 0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHis6-MBP-tev-G-Hs.NRAS(1-169)-ITD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG-NRAS(1-169)-ITD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHis6-MBP-tev-GG-Hs.KRAS4b(2-169)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGG-Hs.KRAS4b(2-169)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29 +/- 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41 +/- 4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHis6-MBP-tev-G-Hs.NRAS(1-169)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG-Hs.NRAS(1-169)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 +/- 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25 +/- 14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHis6-MBP-tev-RAF1(52\u0026ndash;192)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHs.RAF1(52\u0026ndash;192)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 +/- 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51 +/- 22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ldquo; \u0026ldquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHs.RAF1(52\u0026ndash;192) \u003csup\u003e15\u003c/sup\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.6 +/- 0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.4 +/- 0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHis6-MBP-tev-G-Hs.KRAS4b(1-169)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG-Hs.KRAS4b(1-169) D\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8 +/- 0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003e Due to the lack of expressed protein, no scale up was possible.\u003c/p\u003e \u003cp\u003e \u003csup\u003e2\u003c/sup\u003e Due to the poor prognosis at small-scale screen, no scale up was attempted.\u003c/p\u003e \u003cp\u003e \u003csup\u003e3\u003c/sup\u003e Due to the TEV digestion failure of the scale-up prep, no protein was purified.\u003c/p\u003e \u003cp\u003eA similar observation of reduced aggregated protein was made when purifying the G-domain of NRAS (Hs.NRAS(1-169)) which has 92% sequence identity with the KRAS4b G-domain. However, while two IMAC elution peaks were also observed with NRAS (both hosts), as observed with KRAS from \u003cem\u003eE. coli\u003c/em\u003e, the proportion of the \u003cem\u003eV. natriegens\u003c/em\u003e-produced fusion protein recalcitrant to TEV-protease was lower, leading to a significant increase in yield (25 +/- 14 mg/L from \u003cem\u003eV. natriegens\u003c/em\u003e; 6.0 +/- 2 mg/L from \u003cem\u003eE. coli\u003c/em\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese observations led us to attempt to express and purify Hs.RAF1(52\u0026ndash;192), also known as RAF1 CR1, from \u003cem\u003eV. natriegens\u003c/em\u003e. The yield of this protein from \u003cem\u003eE. coli\u003c/em\u003e is also low (less than 10 mg/L) and is a critical reagent in our laboratory. Yields from the \u003cem\u003eV. natriegens\u003c/em\u003e system are significantly higher (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and a Tm can be measured for the protein, which is not possible with the \u003cem\u003eE.\u003c/em\u003e coli produced protein. We also frequently observe that during purification, steps that require concentrating large pools to smaller volumes (e.g. preparing the sample for preparative SEC) take considerably longer (data not shown) for \u003cem\u003eE. coli-\u003c/em\u003eexpressed Hs.RAF1(52\u0026ndash;192).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIsotopic labeling\u003c/h2\u003e \u003cp\u003eA considerable roadblock in our lab is the practical and inexpensive production of isotopically labeled proteins. When purification yields drop below one milligram/liter, the cost of isotopically labeled proteins becomes prohibitive due to the larger fermentation volume (and thus reagent cost). As others have shown recently, \u003cem\u003eV. natriegens\u003c/em\u003e can also be used to produce isotopically labelled proteins [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, we explored producing isotopically labelled Hs.RAF1(52\u0026ndash;192) in \u003cem\u003eV. natriegens\u003c/em\u003e, hoping to reduce costs due to the higher yield. This was investigated by comparing the yield from \u003csup\u003e15\u003c/sup\u003eN isotopic labeling fermentations between \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eV. natriegens\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While modest, the increase from 0.6 mg/L to 1.4 mg/L does merit the consideration of producing these more expensive reagents in the \u003cem\u003eV. natriegens\u003c/em\u003e system. Similarly, we investigated the possibility of producing deuterated proteins in \u003cem\u003eV. natriegens\u003c/em\u003e. As indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, experiments with KRAS4b suggest that \u003cem\u003eV. natriegens\u003c/em\u003e can be considered as an option.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eActivity\u003c/h2\u003e \u003cp\u003eTo assess if the activity of the proteins produced from \u003cem\u003eV. natriegens\u003c/em\u003e was similar to those made from \u003cem\u003eE. coli\u003c/em\u003e, we measured the GTPase activity of KRAS4b and NRAS purified from the two systems by using a phosphate sensor assay. This assay uses the release of phosphate as a surrogate for GTPase activity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Measured rates were similar in both cases: KRAS4b (\u003cem\u003eV. natriegens\u003c/em\u003e 70 \u0026micro;M/min vs \u003cem\u003eE. coli\u003c/em\u003e 83 \u0026micro;M/min); NRAS (\u003cem\u003eV. natriegens\u003c/em\u003e 71 \u0026micro;M/min vs \u003cem\u003eE. coli\u003c/em\u003e 73 \u0026micro;M/min).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe present findings and protocols that should help interested laboratories add \u003cem\u003eV. natriegens\u003c/em\u003e to their recombinant protein production workflow. Specifically, our work defines a set of seed and production media and protocols that builds on our previously published work in \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] (with modifications to Studier\u0026rsquo;s auto-induction studies), that are key to a reproducible system. Additionally, our observations of sensitivity to levels of ampicillin lower than previously reported and the effect Instant Ocean\u0026trade; can have on culture growth, reproducibility, and protein expression, are critical factors to bear in mind. We suspect that at least some of these findings (e.g. ampicillin sensitivity) might be due to differences with the approaches and techniques rather than a difference between isolates of \u003cem\u003eV. natriegens\u003c/em\u003e, but that remains to be investigated.\u003c/p\u003e \u003cp\u003eOnce in place, the potential of the \u003cem\u003eV. natriegens\u003c/em\u003e system, with its fast growth rate, was apparent. The ability to generate a cell pellet within 24 hours from seed culture inoculation, substantially changes the timelines, and thus throughput, of the laboratory (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e): pushing the bottleneck to equipment preparation rather than fermentation. While supplemental oxygen is necessary to achieve this timeline, the improvements in expression, solubility and yield reported here, can still be achieved with extended (~\u0026thinsp;four additional hours) expression at 30\u0026deg;C (data not shown).\u003c/p\u003e \u003cp\u003eOverall, we found using \u003cem\u003eV. natriegens\u003c/em\u003e as an alternative expression host for recombinant protein expression improved several projects in the lab with no increase in cost or effort.\u003c/p\u003e \u003cp\u003eIn some cases, the new expression system was responsible for the success of a protein production project (i.e. the target protein could not be produced from \u003cem\u003eE. coli\u003c/em\u003e, as was the case for AMHR2, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and/or allowed a stalled project to proceed by reducing fermentation volume/costs compared with \u003cem\u003eE. coli\u003c/em\u003e (as was the case for NRAS-ITD from \u003cem\u003eE. coli\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Similarly, increased yield allowed the consideration of isotopically labelled difficult-to-express proteins that would have been prohibitive to produce in \u003cem\u003eE. coli\u003c/em\u003e, as was the case to produce \u003csup\u003e15\u003c/sup\u003eN RAF1(52\u0026ndash;192) and deuterated KRAS4b (both reported here) and \u003csup\u003e13\u003c/sup\u003eC, \u003csup\u003e15\u003c/sup\u003eN, and deuterated RAF1(52\u0026ndash;192) (data not shown).\u003c/p\u003e \u003cp\u003eHowever, the most intriguing aspect of the \u003cem\u003eV. natriegens\u003c/em\u003e system, from our point of view, is the apparent improvement in protein folding suggested by our data. The observations reported here span a range of protein families and expression levels, suggesting the possibility of fundamental differences between \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eV. natriegens\u003c/em\u003e that might be exploited. Whether this is due to increased chaperones, elevated levels of ribosomes, and/or some other factor(s), is not known. These difficult to express proteins, illustrate the potential benefits from adding this easy to use, genetically amenable, and microbiologically diverse \u003cem\u003eV. natriegens\u003c/em\u003e system to the toolbox for recombinant protein production.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cem\u003eV. natriegens\u003c/em\u003e can be an important addition to a protein expression laboratory. While not a replacement for \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eV. natriegens\u003c/em\u003e produces some proteins at higher yield and with less aggregation. Incorporating \u003cem\u003eV. natriegens\u003c/em\u003e into existing small- and large-scale \u003cem\u003eE. coli\u003c/em\u003e project workflows is relatively simple and requires no additional equipment or reagents. In so doing, we have been able to complete several projects heretofore stalled in the standard \u003cem\u003eE. coli\u003c/em\u003e expression system due to poor yield and/or protein aggregation. Given the diversity of proteins represented in these projects from the limited number of protein families we have investigated, is seems likely that \u003cem\u003eV. natriegens\u003c/em\u003e will be useful in the production of additional proteins.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIMAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmobilized metal affinity chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esize exclusion chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;publication\u003c/strong\u003e. Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials.\u003c/strong\u003e All data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u0026nbsp;\u003c/strong\u003eThis work has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract 75N91019D00024. \u0026nbsp; The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVW, CG and JM designed and produced clones. \u0026nbsp;MS, JSH, NR, BH, PVL, JC, and TT carried out expression work. MS, JSH, SM, NR, SP, JM, AJ, PF, MS, MP, SW, J-PD, KS, MD, MH, IP and AC carried out purification of recombinant proteins. \u0026nbsp;MS, JSH, NR, CG, TT, SM, DE and WG conceived and supervised this project and designed experiments. MS, JSH, NR, TT, SM and WG analyzed data. SM and WG wrote the manuscript. All authors edited and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRosano GL, Morales ES, Ceccarelli EA. New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Sci. 2019;28(8):1412\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShilling PJ, Daley DO. Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production. Bio Protoc. 2021;11(16):e4133.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchutz A, et al. 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RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling. J Biol Chem. 2020;295(28):9335\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonsor DA, et al. Structure of the SHOC2-MRAS-PP1C complex provides insights into RAF activation and Noonan syndrome. Nat Struct Mol Biol. 2022;29(10):966\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChao FA, et al. Reduced dynamic complexity allows structure elucidation of an excited state of KRAS(G13D). Commun Biol. 2023;6(1):594.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4178091/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4178091/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: \u003cem\u003eEscherichia coli,\u003c/em\u003e insect cells, and the mammalian cell lines HEK293 and CHO.\u003cem\u003e \u003c/em\u003eRecent interest in \u003cem\u003eVibrio natriegens \u003c/em\u003eas a new prokaryotic recombinant protein expression host is due in part to its short doubling time of \u003cu\u003e\u0026lt;\u003c/u\u003e10 minutes but also stems from the promise of compatibility with techniques and genetic systems developed for \u003cem\u003eE. coli\u003c/em\u003e. We successfully incorporated \u003cem\u003eV. natriegens\u003c/em\u003e as an additional prokaryotic expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in\u003cem\u003e V. natriegens\u003c/em\u003e, we successfully produced several proteins that were difficult or impossible to produce in \u003cem\u003eE. coli\u003c/em\u003e. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with \u003cem\u003eV. natriegens\u003c/em\u003e. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in \u003cem\u003eV. natriegens \u003c/em\u003ethat were impossible and/or unaffordable to produce in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Producing recombinant proteins in Vibrio natriegens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-03 16:59:21","doi":"10.21203/rs.3.rs-4178091/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-15T21:30:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-15T16:46:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-30T15:57:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c012433b-ed72-4ccb-9f56-97fa87d623ef","date":"2024-03-29T14:47:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61826f9d-d647-47ee-8f88-45622a5a9b2c","date":"2024-03-28T20:58:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-28T19:53:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-28T19:30:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-28T19:30:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2024-03-27T19:44:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"784d6131-4b97-4795-b0dc-eeab642d47c1","owner":[],"postedDate":"April 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:05:48+00:00","versionOfRecord":{"articleIdentity":"rs-4178091","link":"https://doi.org/10.1186/s12934-024-02455-5","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2024-07-24 16:15:38","publishedOnDateReadable":"July 24th, 2024"},"versionCreatedAt":"2024-04-03 16:59:21","video":"","vorDoi":"10.1186/s12934-024-02455-5","vorDoiUrl":"https://doi.org/10.1186/s12934-024-02455-5","workflowStages":[]},"version":"v1","identity":"rs-4178091","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4178091","identity":"rs-4178091","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}