Counterintuitive method improves yields of isotopically labeled proteins expressed in flask-cultured Escherichia coli

Counterintuitive method improves yields of isotopically labeled proteins expressed in flask-cultured Escherichia coli | 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 Counterintuitive method improves yields of isotopically labeled proteins expressed in flask-cultured Escherichia coli Miguel Ángel Treviño This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5123333/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Mar, 2025 Read the published version in Journal of Biomolecular NMR → Version 1 posted 9 You are reading this latest preprint version Abstract NMR is a powerful tool for the structural and dynamic study of proteins. One of the necessary conditions for the study of these proteins is their isotopic labeling with 13 C, 15 N and sometimes 2 H. One of the most widely used methods to obtain these labeled proteins is heterologous expression of the proteins in E. coli using 13 C-D-glucose and 15 NH 4 Cl as the sole nutrient sources. In recent years, the price of 13 C-D-glucose has almost tripled, making it essential to develop labeling methods that are as cost effective as possible. In this work, different parameters were studied to achieve the most rational use of 13 C-D-glucose, and an optimized method was developed to obtain labeled proteins with high labeling and low 13 C-D-glucose consumption. Surprisingly, the optimized method is also simple and does not require monitoring of culture growth. Protein expression glucose consumption minimal active time cost reduction 13C labeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION An essential step in studying proteins via NMR is to obtain 13 C-, 15 N- and/or 2 H-labeled proteins at high concentrations to transmit magnetization between different nuclei during NMR experiments. The most widely used method to obtain high quantities of proteins for biological studies is to produce them in heterologous systems, mainly in E. coli , a very well-studied system with many variants and strains. Although biofermentors allow fine, real-time control of many of the variables affecting the growth of bacteria and protein expression, they are expensive, and the majority of laboratories use simpler types of equipment, such as Erlenmeyer flasks or other flasks shaken in incubators. Using this low-tech equipment, enough protein can be obtained in most cases. For unlabeled proteins, the cells are grown in rich medium (LB, 2YT, or other) until the cells grow at an exponential rate, usually at optical densities between 0.6 and 0.8 units. The appropriate inducer is subsequently added to the medium to initiate the expression of the selected protein. In the case of labeled proteins, instead, the classical protocol uses a modification of M9 minimal medium (Miller 1972 ) containing 13 C-D-glucose and 15 NH 4 Cl as the sole carbon and nitrogen sources, respectively, instead of the rich medium, maintaining the point of induction at 0.6–0.8 OD 600 . Over the years, many modifications have been incorporated into this basic protocol to improve the yield of labeled protein due to the high cost of the isotopic sources. Therefore, two main strategies have been followed: 1) modifications to the minimal medium and growth conditions and 2) previous generation of nonlabeled biomass. The first strategy is focused on controlling variables such as the pH of the medium, which usually decreases during bacterial culture. At acidic pH, the cells stop growing and enter a stationary phase, and if the pH is lower than 4.5, further cellular growth can be prevented (Neidhardt et al. 1974 ; Sánchez-Clemente et al. 2018 ). To prevent this effect, the concentrations of buffering substances can be increased to the limit of solubility ((Neidhardt et al. 1974 )(Cai et al. 2019 )). To keep the cells in the exponential growth phase, it is necessary to maintain the bacteria in aerobic conditions, which are usually achieved with a high rate of agitation and/or the use of baffled recipients to generate a turbulent flow of medium instead of a laminar flow. Recently, Cai et al(Cai et al. 2019 ) published a method in which the culture temperature was reduced. This would increase the dissolved oxygen content by approximately 10% when the culture is kept at 30°C or approximately 20% at 25°C compared with the usual 37°C water solubility(Bok et al. 2023 ). In addition, lower growth rates at cooler temperatures increase the number of ribosomes per cell, increasing the proportion of ribosomes available to produce the protein of interest(Marr, 1991 ). With respect to the second strategy, in the seminal paper from Marley et al. (Marley et al. 2001 ),, bacteria were grown in a rich medium to 0.6 OD 600 and then centrifuged, and the medium was changed to a minimal one and the volume was diminished to reduce isotope consumption. After 1 h of adaptation for incorporation of the isotopes and generation of labeled amino acids, protein expression is induced. Variations over this protocol have been proposed. For example, Sivashanmugam et al.(Sivashanmugam et al. 2009 ) grew bacteria up to 3–5 or 5–7 OD 600 in rich media before centrifugation and swapping to minimal medium (in this case, without volume reduction). Like Marley et al., the cells were incubated for 1–2 hours in minimal medium before induction to ensure the incorporation of the isotopes into the precursors of the protein. In both strategies, a fraction of the isotopes is consumed for the generation of biomass (strategy 1) or to ensure complete isotope incorporation and adaptation to the minimal medium (strategy 2), and it is not harnessed for labeled protein generation. In this paper, a method that eliminates the necessity of OD 600 monitoring and centrifugation is presented. This eliminates a stressful step for the bacteria. Additionally, conditions to minimize the nonproductive consumption of isotopes have been studied, increasing the yield of protein without sacrificing isotopic incorporation, which is maintained at approximately 98% for 13 C. MATERIALS and METHODS Plasmids and E. coli strains To analyze each variable, E. coli BL21star(DE3) bacteria transformed with a pET24 plasmid containing the codifying sequence for the human CB1 Cannabinoid Receptor Interacting Protein 1 (CNRIP1) (UniProt Q96F85-1) and containing an Nt-histidine tail and a cleavage site for TEV protease were used. All experiments were carried out with this plasmid/strain To confirm the optimized method, in addition to CNRIP1, a domain of PHOX2b (PHOX2b XS) (Anton et al. (2024)) and complete NEX-XF1 (UniProt O28071) were expressed in BL21star (DE3). Additionally, for the confirmatory experiments, the plasmid encoding CNRIP1 was transformed into the Turner™ (DE3) (Merck, Darmstadt, Germany), C41(DE3) and Shuffle®T7LysY (New England Biolabs, Ipswich, MA) strains. Culture conditions The transformed bacteria were subsequently grown in a slightly modified M9 + + medium (Cai et al.) (Table 1) in Erlenmeyer flasks or Tunair flasks (IBI Scientific, Dubuque, IA) with capacities at least 10 times greater than the medium volume. To generate inocula, the transformed strain was grown in LB overnight at 37°C. The grown inoculum was directly added to the minimal medium. The cultures were composed of three phases: 1) biomass generation in minimal media with nonlabeled glucose, 2) addition of extra glucose (labeled or unlabeled, depending on the tested conditions) for the biosynthesis of amino acids and culture for the incorporation of isotopes, and 3) induction and protein expression. The temperatures and times in each step were 25°C overnight (phase 1), 30°C for a variable time (phase 2) and 20°C for 24 hours (phase 3), except when indicated. Cultures for other methods, for comparison, were performed as described in their original papers (Cai et al ( 2019 ), Sivashanmugam et al ( 2009 ), Marley et al ( 2001 )). Culture Variables Determination The optical density was measured at 600 nm with a Nanodrop One spectrophotometer (Thermo Scientific, Waltham, MA). The free D-glucose concentration in the media was determined with a commercial QuantiChrom glucose assay kit (Bioassay Systems, Hayward, CA), which generates blue color due to the formation of an imino bond between the aldehyde group for sugars and o-toluidine. After a centrifugation pulse of the culture to eliminate the bacteria, 2 to 5 microliters of the supernatant were mixed with 50 µl of the commercial reactive, following the manufacturer’s instructions. A 630 measurements were performed with a Nanodrop One spectrophotometer, and the D-glucose concentration was calculated by interpolating in calibration curves obtained during the same experiment. Expressed Protein Quantification Volumes corresponding to equivalent amounts of D-glucose added during phase 2 (i.e., 100 µL for 1% glucose added, 200 µL for 0.5% glucose added or 250 µL for 0.4% glucose) of the final expression cultures were centrifuged, and the pellets were lysed with 50 µL of bugbuster (Merck, Rahway, NJ). After centrifugation, the insoluble fractions were solubilized in 50 µL of 8 M urea. Both fractions were pooled and mixed with the same volume of loading buffer for PAGE. Two to 10 µL samples were loaded in a custom gradient (4–25%) acrylamide gel containing 3.75% trichloroethanol for direct fluorescence detection of triptophans (Kazmin et al. 2002 ), and the proteins were separated by PAGE. Each sample was loaded at least 3 times. Bands were quantified in a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA) using the free stain option and analyzed with ImageLab software. Protein purification For mass spectrometry experiments, bacteria from 5 ml of growth cultures were centrifuged and resuspended in 1 mL of 50 mM potassium phosphate (pH 8), 300 mM NaCl, and 10 mM imidazole with 1 µL of Halt inhibitors (Thermo Scientific, Waltham, MA). The cells were sonicated, and the lysate was centrifuged. One hundred microliters of nickel high-density beads (Agarose Bead Technologies, Torrejón de Ardoz, Spain) were added, and the mixture was loaded onto a MicroBiospin empty column (Bio-Rad, Hercules, CA). After washing with the same buffer, CNRIP1 was eluted in 400 µL of 50 mM potassium phosphate (pH 8), 300 mM NaCl, and 500 mM imidazole. Two micrograms of TEV protease were added, and the sample was dialyzed against 1 L of 5 mM potassium phosphate, (pH6.8), 10 mM NaCl, and 1 mM β-mercaptoethanol. For NMR spectroscopy, the cultures were scaled to 50 or 100 mL. Lysis was performed analogously, but the lysate supernatants were loaded in HisTrap 5 mL FF columns (Cytiva, Marlborough, MA). The eluates were dialyzed against 5 mM potassium phosphate (pH8), 10 mM NaCl, and simultaneously cleaved with TEV protease. The dialyzed samples were loaded in the same column at the flowthrough collected and redyalized. The samples were then loaded onto HiTrap 1 mL SP columns (Cytiva, Marlborough, MA). The samples were prepared in 5 mM potassium phosphate (pH 6.8) and 10 mM NaCl. Mass spectrometry The mass of the purified proteins was determined by mass spectrometry. The samples were analyzed in an HPLC 1100 Series LC System (Agilent Technologies, Palo Alto, USA) coupled to an HTC-Ultra ETD II ion trap mass spectrometer (Bruker Daltonics, Fremont, USA) with an electrospray ionization (ESI) source. The molecular masses of the proteins were calculated by deconvolution of the ESI‒MS spectra using the Thermo Finningan BIOMASSTM software (Thermo Fisher Scientific, San José, CA, USA). NMR spectroscopy 1D 1 H-spectra and 1 H- 13 C-HSQC spectra were recorded on a Bruker Avance Neo 800 MHz ( 1 H) spectrometer fitted with a cryoprobe and z-gradients. The experiments were collected at 25°C. For coupled spectra, the same experiments were performed as for conventional decoupled spectra, but no 13 C decoupling pulses were applied during acquisition. RESULTS and DISCUSSION Previous work (Cai et al. 2019) has shown that in E. coli cultures at low temperatures, up to OD 600 = 6, before induction, a high amount of protein is obtained (relative to the amount of medium used), with isotopic labeling of approximately 97%. Despite this good result, there is a percentage of the 13 C-D-glucose that is used just to generate biomass and therefore it is “wasted” to improve the yield of labeled protein. A simple way to improve this would be to combine this protocol with a first step of biomass generation in rich media, similar to other protocols, which then switch to labeled media by centrifugation and produce high yields of labeled proteins. One drawback of this approximation is that centrifugation steps can be stressful for the bacteria, and they recover slowly. In fact, the OD 600 can drop in the first few moments in minimal media, and it is difficult to estimate how long the bacteria need to be grown in these media before induction to maximize expression and minimize detrimental unproductive consumption of labeled nutrients. Therefore, it can be hypothesized that the complete consumption of unlabeled glucose in minimal media could be as efficient in terms of biomass production as the use of rich media but avoid the stress of centrifugation. It has been reported that E. coli recover quickly from short periods of starvation with no apparent sequelae (Lempp et al. 2019). In addition, the cells adapt to grow in these minimal media from the beginning, further reducing the stress of switching from rich to poor media and minimizing the time required to incorporate labeled metabolites. To evaluate this hypothesis, biomass production and glucose consumption were monitored under different conditions (Fig. 1). Different initial D-glucose concentrations were tested. In all the samples, after 23 h of growth at 25°C, 0.5% D-glucose was added, and the culture continued to grow at 30°C for 1.5 h, followed by growth at 20°C for another 24 h. The depletion of glucose after overnight growth was complete under all the conditions tested, and the growth rate recovery appeared to be complete after the addition of supplemental 0.5% D-glucose. Although it was predictable that some of this additional D-glucose would be consumed during the isotope integration step, after 1.5 h at 30°C, the remaining nutrient content was extremely low for the cultures with high initial glucose concentrations, leaving less than 0.1% D-glucose available for the protein expression step under the initial 0.5% D-glucose condition, and after 3 additional hours at 20°C, no glucose remained for 0.3, 0.4 or 0.5% initial glucose conditions. Thus, a counterintuitive result was found: it is not convenient to produce large amounts of biomass but rather to find a compromise between biomass and the consumption of labeled glucose to improve the final protein yield. The second variable monitored was the incorporation of 13 C into the labeled protein. The data show that for high initial glucose, only partial incorporation into the protein was reached, but when the initial glucose was reduced from 0.5–0.4% and to 0.2%, the incorporation increased from 75–90% and 98%, respectively, as detected by NMR (Fig. 2). These results led to the testing of other conditions - decreasing the glucose in the biomass generation step and increasing the labeled glucose added in the “isotope integration step” -. The protein yield under each new condition was greater for 0.2–0.3% D-glucose in the “biomass generation step” combined with 1% D-glucose added in the “isotope integration step” (Fig. 3). Considering the data from the previous experiments, the effect of varying the time of the "isotope integration step" was tested by monitoring the protein yield and 13 C incorporation (Fig. 4). A second counterintuitive fact appeared: there is a minimal effect of the length of this phase on 13 C incorporation, which is always approximately 97–98% and can even be eliminated. As the initial data indicated that for 0.5/0.5% conditions, even with a 1.5 h isotope integration step, the incorporation was approximately 75% it seems that in any case, the relative ratio between the initial unlabeled and subsequently added labeled glucose should not exceed 20% (0.2% initial D-glucose, 1% labeled D-glucose). Although all biomass generation cultures were performed at 25°C to ensure that maximum O 2 was available to the cells, the influence of temperature on growth was monitored at 25°C, 30°C and 37°C (Fig. 5). Complete glucose depletion is reached in overnight cultures at 25°C or 30°C, whereas at 37°C, complete glucose depletion is achieved after approximately 5.5 hours. This would allow the biomass generation step to be shortened. However, the high growth rate at 37°C could have disadvantages, such as a lower number of ribosomes per cell (Marr 1991) or a possible microaerobic state, which could promote the accumulation of acetate (Partridge et al. 2007) and therefore inhibit cellular growth or the expression of the desired protein (Shiloach and Fass 2005). Finally, other E. coli strains or plasmid‒strain combinations may be less efficient in nutrient consumption than those tested here and may require longer culture times. Although the total time for 25°C or 30°C cultures is longer, the active time for the present method decreases from approximately 1–2 hours (due to measurements of the OD 600 until it reaches 0.6, centrifugation, resuspension in minimal medium, and addition of inductor) to approximately 5–10 minutes (due to the addition of labeled nutrients and inductor simultaneously). Taking in consideration all these facts, to grow the culture at 25ºC is recommended for this method. Although we have not tested the possibility of starting the culture directly from colonies from a plate instead of an overnight liquid preinoculum culture, as previously reported (Sivashanmugam et al. 2009), it could also diminish the total time of culture in addition to the already diminished active time. The influence of different glucose concentrations during the expression step on yield was also investigated (Fig. 6). A slight increase in yield was detected when the glucose concentration increased from 1–1.5%, but the yield decreased at higher percentages. In any case, the increments are in the range of error, so there is no evident advantage in the use of higher glucose percentages. In any case, this method clearly improves the yield of labeled protein compared with some of the previously described protocols, and even in the least favorable case, there is a 20% increase. The tests indicate that 50 mL of culture (0.5 g of 13 C-D-glucose) is sufficient to obtain 10–20 mg of purified labeled protein. Notably, Sivashanmugam's protocol was modified, and 3 times more NH4Cl was used for this experiment. When the original amounts were used, the yield was drastically reduced (see Fig. 8 below), indicating the importance of an appropriate ratio of nutrients to obtain the maximum yield in any protocol. The ratios of NH4Cl and D-glucose in this recipe were calculated to ensure that glucose was the limiting nutrient. In this way, it can be ensured that all the unlabeled glucose has been depleted in the biomass production step. Since 13 C-labeled proteins for NMR are usually also 15 N-labeled, 15 NH4Cl must be used from the beginning because it cannot be assured that there is no remaining nitrogen that has not been consumed in the initial steps. Since 15 NH4Cl is much cheaper than 13 C-D-glucose, net savings still prevail, and it will be even more significant if we need to use D7- 13 C-D-glucose or other isotope-labeled precursors, which are even more expensive than 13 C-D-glucose. Finally, two experiments were carried out to test whether this method could be used universally, regardless of the strain or protein expressed. To check for strain independence, CNRP1 was expressed in the BL21star(DE3), Turner™(DE3), C41(DE3), and Shuffle® T7 LysY strains, and 13 C incorporation was determined by mass spectrometry. No differences in glucose uptake or protein expression were found among these strains and the 13 C incorporation was almost identical (97.6%, 97.4%, 97.9% and 96.9%, respectively) (Fig. 7). To determine the variations of yield in the expression of different proteins depending of the protocol used, the method was tested for proteins CNRIP1, PHOX2b XS and NEX XF1 (Fig. 8). In all of them, a higher quantity of protein was obtained with the method described here than with the other protocols. Notably, the ratios between the different methods are not exactly the same for the 3 proteins, but in any case, the yield per gram of glucose is higher with this method for the three proteins tested. CONCLUSIONS A new method for the production of 13 C-labeled proteins has been developed (a detailed protocol is provided in the supplemental material), which minimizes the time commitment required (Fig. 9 ) and optimizes the consumption of labeled nutrients. An initial culture step at 25–30°C in minimal medium allows biomass to be generated but avoids centrifugation. No "isotope integration step" is necessary, and simply adjusting the temperature to the appropriate level for protein expression before adding labeled nutrients and the inductor is enough to obtain maximum protein yields with high 13 C incorporation. This protocol, with minimal variations, could be used to generate other labels, such as selenomethionine for X-ray crystallography and for the selective labeling of different amino acid positions using 1,3- 13 C-glycerol or 2- 13 C-glycerol or other precursors as carbon sources. As well as for triple ( 2 H, 15 N, 13 C) labeled proteins. In summary, two unexpected and counterintuitive findings, the limited biomass generation and the irrelevance of the isotope integration step, have allowed to develop a highly optimized protocol that offers significant advantages over other published protocols: simplicity, no need for monitoring, minimal active time, high yields of labeled protein and cost reduction. Declarations Author Contribution M.A.T. to the study conception and design the study, made the experiments and wrote the manuscript.. Material preparation, data collection and analysis were performed by [full name], [full name] and [full name]. The first draft of the manuscript was written by [full name] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement The author thanks Daniel Calvo for technical assistance, David Pantoja-Uceda for assistance with NMR spectroscopy, Plácido Galindo from the mass spectrometry data adquisition, and Douglas V. Laurents for constructive feedback. Proteins were expressed, and NMR spectra were recorded at the Manuel Rico NMR Laboratory (LMR), a node of the ICTS for biomolecular NMR (R-LRB). References Antón R, Treviño MÁ, Pantoja-Uceda D, Félix S, Babu M, Cabrita EJ, Zweckstetter M, Tinnefeld P, Vera AM, Oroz J (2024) Alternative low-populated conformations prompt phase transitions in polyalanine repeat expansions. Nature Communications 15: 1925. https://doi.org/10.1038/s41467-024-46236-5 Bok F, Moog HC, Brendler V (2023) The solubility of oxygen in water and saline solutions. Front Nuclear Eng 2:1292254. https://doi.org/10.3389/fnuen.2023.1158109 Cai M, Huang Y, Craigie R, Clore GM (2019) A simple protocol for expression of isotope-labeled proteins in Escherichia coli grown in shaker flasks at high cell density. J Biomol NMR 73:743–748. https://doi.org/10.1007/s10858-019-00285-x Kazmin D, Edwards RA, Turner RJ et al (2002) Visualization of proteins in acrylamide gels using ultraviolet illumination. Anal Biochem 301:91–96. https://doi.org/10.1006/abio.2001.5488 Lempp M, Farke N, Kuntz M et al (2019) Systematic identification of metabolites controlling gene expression in E. coli. Nat Commun 10:4463. https://doi.org/10.1038/s41467-019-12474-1 Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75. https://doi.org/10.1023/A:1011254402785 Marr AG (1991) Growth rate of Escherichia coli. Microbiol Rev 55:316–333. https://doi.org/10.1128/mr.55.2.316-333.1991 Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor,NY Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747. https://doi.org/10.1128/jb.119.3.736-747.1974 Partridge JD, Sanguinetti G, Dibden DP et al (2007) Transition of Escherichia coli from aerobic to micro-aerobic conditions involves fast and slow reacting regulatory components. J Biol Chem 282:11230–11237. https://doi.org/10.1074/jbc.M700728200 Sánchez-Clemente R, Igeño MI, Población AG et al (2018) Study of pH Changes in Media during Bacterial Growth of Several Environmental Strains. Proceedings 2: 1297. https://doi.org/10.3390/proceedings2201297 Shiloach J, Fass R (2005) Growing E. coli to high cell density - A historical perspective on method development. Biotechnol Adv 23:345–357. https://doi.org/10.1016/j.biotechadv.2005.04.004 Sivashanmugam A, Murray V, Cui C et al (2009) Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci 18:936–948. https://doi.org/10.1002/pro.102 Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTALMATERIALmod.docx Cite Share Download PDF Status: Published Journal Publication published 01 Mar, 2025 Read the published version in Journal of Biomolecular NMR → Version 1 posted Editorial decision: Revision requested 02 Dec, 2024 Reviews received at journal 27 Nov, 2024 Reviews received at journal 21 Nov, 2024 Reviewers agreed at journal 14 Nov, 2024 Reviewers agreed at journal 04 Nov, 2024 Reviewers invited by journal 21 Oct, 2024 Editor assigned by journal 14 Oct, 2024 Submission checks completed at journal 21 Sep, 2024 First submitted to journal 20 Sep, 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. 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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-5123333","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":385203272,"identity":"585114ee-cfd1-40ff-a954-b0122279c5cd","order_by":0,"name":"Miguel Ángel Treviño","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie3PsQrCMBCA4SuBdAlkrSB9AyESqKOvUpfOupcS6OAkrm6+giI4Fw7qSzgoBeeAk+BgUh0cJNXNIf+SDPeRC4DP97cJAE4AtLmy7mn6Ir0SgtUPxKrqSbobrBfnhk2PsTyQrYK86HNFULtIUodSMnGRCdKZghpZVNEschNKeyuBkz2y0SlQFRPAEudihoQ3S3YlG6pAFYbwa8dilII2ZENaQuwr0LFYRiJDZGT/ktq/IE3cBOtAp3eM+RK3SufFmM/LxrnYewJSe5Bv51vi8/l8vk89AEsgQfYaZ4tEAAAAAElFTkSuQmCC","orcid":"","institution":"Instituto de Química Física Blas Cabrera, Consejo Superior de Investigaciones Científicas","correspondingAuthor":true,"prefix":"","firstName":"Miguel","middleName":"Ángel","lastName":"Treviño","suffix":""}],"badges":[],"createdAt":"2024-09-20 11:38:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5123333/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5123333/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10858-025-00461-2","type":"published","date":"2025-03-01T15:58:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74435167,"identity":"b9a40f70-757f-4e01-9c8b-c04c011eaa91","added_by":"auto","created_at":"2025-01-22 09:22:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116944,"visible":true,"origin":"","legend":"\u003cp\u003eOptical density (top) and dissolved D-glucose (bottom) in \u003cem\u003eE. coli\u003c/em\u003e cultures as a function of initial D-glucose concentration. Cultures were kept at the temperatures indicated at the top of the figure, mimicking the steps in the induced cultures, i.e., 25°C during the 'biomass generation step', 30°C after the addition of 0.5% D-glucose to simulate the 'isotope integration step' and 20°C to simulate growth after the addition of IPTG in the 'expression step'. The dashed lines do not represent linear growth and are only added to help locate points from the same conditions.\u003c/p\u003e","description":"","filename":"Fig11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/2589996394836079c2237700.jpg"},{"id":74435173,"identity":"965eb616-25a3-498c-97ad-cf3fe695d07e","added_by":"auto","created_at":"2025-01-22 09:22:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":425469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC incorporation under different culture conditions measured by NMR. Spectra with or without a \u003csup\u003e13\u003c/sup\u003eC decoupling pulse are shown. On the left, the full spectra are shown; on the right, the selected region is magnified. To improve clarity, the spectra are shifted vertically.\u003c/p\u003e","description":"","filename":"Fig12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/88036edb40a774ef94c99637.jpg"},{"id":74436604,"identity":"e547fa3b-7ce4-426e-a25c-023ce0ebdda4","added_by":"auto","created_at":"2025-01-22 09:30:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36491,"visible":true,"origin":"","legend":"\u003cp\u003eYields of protein with different percentages of initial D-glucose and D-glucose added.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Fluorescence emission under UV exposure of an SDS‒PAGE gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e: Bar graph representing the relative fluorescence according to the method of Marley et al. considered as a unit.\u003c/p\u003e","description":"","filename":"Fig13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/584109edc30f9a508133a4cc.jpg"},{"id":74436605,"identity":"494a6e76-eb08-4751-bcfa-621a5c60e97a","added_by":"auto","created_at":"2025-01-22 09:30:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":133203,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different \"isotope integration\" times on yield and \u003csup\u003e13\u003c/sup\u003eC labeling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Fluorescence emission under UV exposure of an SDS‒PAGE gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e: Bar graph representing the relative fluorescence according to the method of Marley et al. 2009, which was used as a unit. The data are the mean of three experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e: Mass spectra. The masses of the more intense peak for each condition are given in the square. The expected mass for CNRIP1a without labeling is 18705 Da. To calculate percentage of incorporation the formula % incorporation=100x (experimental result-theoretical mass without labeling)/(theoretical mass at 100% labeling-theoretical mass without labeling) can be used. So, for masses of 19527 and 19528 Da, % incorporation was 97.5 and 97.6%, respectively.\u003c/p\u003e","description":"","filename":"Fig14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/b519d90d3922b144eed52555.jpg"},{"id":74435169,"identity":"e829236b-2b68-4744-9356-aaef2897e9ff","added_by":"auto","created_at":"2025-01-22 09:22:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":64180,"visible":true,"origin":"","legend":"\u003cp\u003eOptical density (top) and dissolved D-glucose (bottom) in \u003cem\u003eE. coli\u003c/em\u003e BL21star(DE3) cultures as a function of temperature. The dashed lines do not represent linear growth and are only added to make it easier to locate symbols from the same conditions. At 30°C, no recording was made at the likely hour of complete D-glucose consumption, so an unfilled circle was added on the basis of the exponential behavior of the curves to provide an indication of the approximate time of the event.\u003c/p\u003e","description":"","filename":"Fig15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/0c82f7c197aba1b27062bb1c.jpg"},{"id":74435175,"identity":"86e42bc4-0798-478b-9d59-9fc6625eb98a","added_by":"auto","created_at":"2025-01-22 09:22:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":247824,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the percentage of D-glucose added in the \"protein expression step\" on protein yield. a: Fluorescence emission under UV exposure of an SDS‒PAGE gel. b: Bar graph of relative fluorescence of the band corresponding to CNRIP1. Band intensities were referenced to the HCDII band, a modification of Sivashanmugam et al. (2009) with 3 times more NH4Cl than the original. For comparison, a checkered bar corresponding to the relative yield obtained using Marley et al. protocol, which was determined in previous experiments, was added to the graph. The data are from five experiments.\u003c/p\u003e","description":"","filename":"Fig16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/790b1661345eb58e8d109e47.jpg"},{"id":74435177,"identity":"77f8dcb0-271d-4883-abc3-fe18acbf4270","added_by":"auto","created_at":"2025-01-22 09:22:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":160929,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the \u003cem\u003eE. coli\u003c/em\u003e strain on glucose consumption and protein expression. Top: dissolved glucose at different culture times for 3 different \u003cem\u003eE. coli\u003c/em\u003estrains. Bottom: mass spectra of CNRIP1a expressed in different strains with the method described in this paper. The mass of the more intense peak for each condition is given in the square. To calculate percentage of incorporation the formula % incorporation=100x (experimental result-theoretical mass without labeling)/(theoretical mass at 100% labeling-theoretical mass without labeling) can be used. So, for masses of 19522, 19525, 19528, and 19530 Da, % incorporation was 96.9%, 97.3%, 97.6%, and 97.9%respectively. The second intense peak detected for some strains corresponded to the double ionized DNaseI added during purification.\u003c/p\u003e","description":"","filename":"Fig17.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/fa25433bb19943633f12058a.jpg"},{"id":74435172,"identity":"65e6e70c-e699-458d-885e-bca633228ba9","added_by":"auto","created_at":"2025-01-22 09:22:22","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":82443,"visible":true,"origin":"","legend":"\u003cp\u003eRelative protein yield for three different proteins using different protocols. Each protein expression is referenced to the expression obtained using Marley’s protocol, which is considered a unit.\u003c/p\u003e","description":"","filename":"Fig18.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/1c11d314b66dbf29b80a15d4.jpg"},{"id":74435174,"identity":"3f2f6668-3da5-43b0-89b8-00ce0e1a9616","added_by":"auto","created_at":"2025-01-22 09:22:22","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":232716,"visible":true,"origin":"","legend":"\u003cp\u003eOutline of some protein expression methods compared with the one described here. Different OD\u003csub\u003e600\u003c/sub\u003e measurements may be required for these methods, increasing the required active time\u003c/p\u003e","description":"","filename":"Fig19.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/24610ac2e51bf451926420ef.jpg"},{"id":77622582,"identity":"7a6b41f3-7910-47d9-aa7d-fc6f232e68e9","added_by":"auto","created_at":"2025-03-03 16:08:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2007871,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/f46113a7-4269-4c47-9bb6-b5fa2ccb5c80.pdf"},{"id":74435171,"identity":"76593289-a36f-40aa-bdb4-a886f1a1fda8","added_by":"auto","created_at":"2025-01-22 09:22:22","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":20779,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTALMATERIALmod.docx","url":"https://assets-eu.researchsquare.com/files/rs-5123333/v1/68972d52771a1a6be49e50ca.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Counterintuitive method improves yields of isotopically labeled proteins expressed in flask-cultured Escherichia coli","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAn essential step in studying proteins via NMR is to obtain \u003csup\u003e13\u003c/sup\u003eC-, \u003csup\u003e15\u003c/sup\u003eN- and/or \u003csup\u003e2\u003c/sup\u003eH-labeled proteins at high concentrations to transmit magnetization between different nuclei during NMR experiments.\u003c/p\u003e \u003cp\u003eThe most widely used method to obtain high quantities of proteins for biological studies is to produce them in heterologous systems, mainly in \u003cem\u003eE. coli\u003c/em\u003e, a very well-studied system with many variants and strains. Although biofermentors allow fine, real-time control of many of the variables affecting the growth of bacteria and protein expression, they are expensive, and the majority of laboratories use simpler types of equipment, such as Erlenmeyer flasks or other flasks shaken in incubators. Using this low-tech equipment, enough protein can be obtained in most cases. For unlabeled proteins, the cells are grown in rich medium (LB, 2YT, or other) until the cells grow at an exponential rate, usually at optical densities between 0.6 and 0.8 units. The appropriate inducer is subsequently added to the medium to initiate the expression of the selected protein.\u003c/p\u003e \u003cp\u003eIn the case of labeled proteins, instead, the classical protocol uses a modification of M9 minimal medium (Miller \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1972\u003c/span\u003e) containing \u003csup\u003e13\u003c/sup\u003eC-D-glucose and \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl as the sole carbon and nitrogen sources, respectively, instead of the rich medium, maintaining the point of induction at 0.6\u0026ndash;0.8 OD\u003csub\u003e600\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eOver the years, many modifications have been incorporated into this basic protocol to improve the yield of labeled protein due to the high cost of the isotopic sources. Therefore, two main strategies have been followed: 1) modifications to the minimal medium and growth conditions and 2) previous generation of nonlabeled biomass.\u003c/p\u003e \u003cp\u003eThe first strategy is focused on controlling variables such as the pH of the medium, which usually decreases during bacterial culture. At acidic pH, the cells stop growing and enter a stationary phase, and if the pH is lower than 4.5, further cellular growth can be prevented (Neidhardt et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; S\u0026aacute;nchez-Clemente et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To prevent this effect, the concentrations of buffering substances can be increased to the limit of solubility ((Neidhardt et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1974\u003c/span\u003e)(Cai et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)). To keep the cells in the exponential growth phase, it is necessary to maintain the bacteria in aerobic conditions, which are usually achieved with a high rate of agitation and/or the use of baffled recipients to generate a turbulent flow of medium instead of a laminar flow. Recently, Cai et al(Cai et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) published a method in which the culture temperature was reduced. This would increase the dissolved oxygen content by approximately 10% when the culture is kept at 30\u0026deg;C or approximately 20% at 25\u0026deg;C compared with the usual 37\u0026deg;C water solubility(Bok et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, lower growth rates at cooler temperatures increase the number of ribosomes per cell, increasing the proportion of ribosomes available to produce the protein of interest(Marr, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWith respect to the second strategy, in the seminal paper from Marley et al. (Marley et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e),, bacteria were grown in a rich medium to 0.6 OD\u003csub\u003e600\u003c/sub\u003e and then centrifuged, and the medium was changed to a minimal one and the volume was diminished to reduce isotope consumption. After 1 h of adaptation for incorporation of the isotopes and generation of labeled amino acids, protein expression is induced. Variations over this protocol have been proposed. For example, Sivashanmugam et al.(Sivashanmugam et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) grew bacteria up to 3\u0026ndash;5 or 5\u0026ndash;7 OD\u003csub\u003e600\u003c/sub\u003e in rich media before centrifugation and swapping to minimal medium (in this case, without volume reduction). Like Marley et al., the cells were incubated for 1\u0026ndash;2 hours in minimal medium before induction to ensure the incorporation of the isotopes into the precursors of the protein.\u003c/p\u003e \u003cp\u003eIn both strategies, a fraction of the isotopes is consumed for the generation of biomass (strategy 1) or to ensure complete isotope incorporation and adaptation to the minimal medium (strategy 2), and it is not harnessed for labeled protein generation.\u003c/p\u003e \u003cp\u003eIn this paper, a method that eliminates the necessity of OD\u003csub\u003e600\u003c/sub\u003e monitoring and centrifugation is presented. This eliminates a stressful step for the bacteria. Additionally, conditions to minimize the nonproductive consumption of isotopes have been studied, increasing the yield of protein without sacrificing isotopic incorporation, which is maintained at approximately 98% for \u003csup\u003e13\u003c/sup\u003eC.\u003c/p\u003e"},{"header":"MATERIALS and METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids and E. coli strains\u003c/h2\u003e \u003cp\u003eTo analyze each variable, \u003cem\u003eE. coli\u003c/em\u003e BL21star(DE3) bacteria transformed with a pET24 plasmid containing the codifying sequence for the human CB1 Cannabinoid Receptor Interacting Protein 1 (CNRIP1) (UniProt Q96F85-1) and containing an Nt-histidine tail and a cleavage site for TEV protease were used. All experiments were carried out with this plasmid/strain\u003c/p\u003e \u003cp\u003eTo confirm the optimized method, in addition to CNRIP1, a domain of PHOX2b (PHOX2b XS) (Anton et al. (2024)) and complete NEX-XF1 (UniProt O28071) were expressed in BL21star (DE3). Additionally, for the confirmatory experiments, the plasmid encoding CNRIP1 was transformed into the Turner\u0026trade; (DE3) (Merck, Darmstadt, Germany), C41(DE3) and Shuffle\u0026reg;T7LysY (New England Biolabs, Ipswich, MA) strains.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCulture conditions\u003c/h3\u003e\n\u003cp\u003eThe transformed bacteria were subsequently grown in a slightly modified M9\u0026thinsp;+\u0026thinsp;+\u0026thinsp;medium (Cai et al.) (Table\u0026nbsp;1) in Erlenmeyer flasks or Tunair flasks (IBI Scientific, Dubuque, IA) with capacities at least 10 times greater than the medium volume. To generate inocula, the transformed strain was grown in LB overnight at 37\u0026deg;C. The grown inoculum was directly added to the minimal medium.\u003c/p\u003e \u003cp\u003e\u003cimg 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\u003cp\u003eThe cultures were composed of three phases: 1) biomass generation in minimal media with nonlabeled glucose, 2) addition of extra glucose (labeled or unlabeled, depending on the tested conditions) for the biosynthesis of amino acids and culture for the incorporation of isotopes, and 3) induction and protein expression.\u003c/p\u003e \u003cp\u003eThe temperatures and times in each step were 25\u0026deg;C overnight (phase 1), 30\u0026deg;C for a variable time (phase 2) and 20\u0026deg;C for 24 hours (phase 3), except when indicated.\u003c/p\u003e \u003cp\u003eCultures for other methods, for comparison, were performed as described in their original papers (Cai et al (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), Sivashanmugam et al (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), Marley et al (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e)).\u003c/p\u003e\n\u003ch3\u003eCulture Variables Determination\u003c/h3\u003e\n\u003cp\u003eThe optical density was measured at 600 nm with a Nanodrop One spectrophotometer (Thermo Scientific, Waltham, MA).\u003c/p\u003e \u003cp\u003eThe free D-glucose concentration in the media was determined with a commercial QuantiChrom glucose assay kit (Bioassay Systems, Hayward, CA), which generates blue color due to the formation of an imino bond between the aldehyde group for sugars and o-toluidine. After a centrifugation pulse of the culture to eliminate the bacteria, 2 to 5 microliters of the supernatant were mixed with 50 \u0026micro;l of the commercial reactive, following the manufacturer\u0026rsquo;s instructions. A\u003csub\u003e630\u003c/sub\u003e measurements were performed with a Nanodrop One spectrophotometer, and the D-glucose concentration was calculated by interpolating in calibration curves obtained during the same experiment.\u003c/p\u003e\n\u003ch3\u003eExpressed Protein Quantification\u003c/h3\u003e\n\u003cp\u003eVolumes corresponding to equivalent amounts of D-glucose added during phase 2 (i.e., 100 \u0026micro;L for 1% glucose added, 200 \u0026micro;L for 0.5% glucose added or 250 \u0026micro;L for 0.4% glucose) of the final expression cultures were centrifuged, and the pellets were lysed with 50 \u0026micro;L of bugbuster (Merck, Rahway, NJ). After centrifugation, the insoluble fractions were solubilized in 50 \u0026micro;L of 8 M urea. Both fractions were pooled and mixed with the same volume of loading buffer for PAGE. Two to 10 \u0026micro;L samples were loaded in a custom gradient (4\u0026ndash;25%) acrylamide gel containing 3.75% trichloroethanol for direct fluorescence detection of triptophans (Kazmin et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and the proteins were separated by PAGE. Each sample was loaded at least 3 times.\u003c/p\u003e \u003cp\u003eBands were quantified in a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA) using the free stain option and analyzed with ImageLab software.\u003c/p\u003e\n\u003ch3\u003eProtein purification\u003c/h3\u003e\n\u003cp\u003eFor mass spectrometry experiments, bacteria from 5 ml of growth cultures were centrifuged and resuspended in 1 mL of 50 mM potassium phosphate (pH 8), 300 mM NaCl, and 10 mM imidazole with 1 \u0026micro;L of Halt inhibitors (Thermo Scientific, Waltham, MA). The cells were sonicated, and the lysate was centrifuged. One hundred microliters of nickel high-density beads (Agarose Bead Technologies, Torrej\u0026oacute;n de Ardoz, Spain) were added, and the mixture was loaded onto a MicroBiospin empty column (Bio-Rad, Hercules, CA). After washing with the same buffer, CNRIP1 was eluted in 400 \u0026micro;L of 50 mM potassium phosphate (pH 8), 300 mM NaCl, and 500 mM imidazole. Two micrograms of TEV protease were added, and the sample was dialyzed against 1 L of 5 mM potassium phosphate, (pH6.8), 10 mM NaCl, and 1 mM β-mercaptoethanol.\u003c/p\u003e \u003cp\u003eFor NMR spectroscopy, the cultures were scaled to 50 or 100 mL. Lysis was performed analogously, but the lysate supernatants were loaded in HisTrap 5 mL FF columns (Cytiva, Marlborough, MA). The eluates were dialyzed against 5 mM potassium phosphate (pH8), 10 mM NaCl, and simultaneously cleaved with TEV protease. The dialyzed samples were loaded in the same column at the flowthrough collected and redyalized. The samples were then loaded onto HiTrap 1 mL SP columns (Cytiva, Marlborough, MA). The samples were prepared in 5 mM potassium phosphate (pH 6.8) and 10 mM NaCl.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry\u003c/h2\u003e \u003cp\u003eThe mass of the purified proteins was determined by mass spectrometry. The samples were analyzed in an HPLC 1100 Series LC System (Agilent Technologies, Palo Alto, USA) coupled to an HTC-Ultra ETD II ion trap mass spectrometer (Bruker Daltonics, Fremont, USA) with an electrospray ionization (ESI) source. The molecular masses of the proteins were calculated by deconvolution of the ESI‒MS spectra using the Thermo Finningan BIOMASSTM software (Thermo Fisher Scientific, San Jos\u0026eacute;, CA, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNMR spectroscopy\u003c/h3\u003e\n\u003cp\u003e1D \u003csup\u003e1\u003c/sup\u003eH-spectra and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC-HSQC spectra were recorded on a Bruker Avance Neo 800 MHz (\u003csup\u003e1\u003c/sup\u003eH) spectrometer fitted with a cryoprobe and z-gradients. The experiments were collected at 25\u0026deg;C.\u003c/p\u003e \u003cp\u003eFor coupled spectra, the same experiments were performed as for conventional decoupled spectra, but no \u003csup\u003e13\u003c/sup\u003eC decoupling pulses were applied during acquisition.\u003c/p\u003e"},{"header":"RESULTS and DISCUSSION","content":"\u003cp\u003ePrevious work (Cai et al. 2019) has shown that in \u003cem\u003eE. coli\u003c/em\u003e cultures at low temperatures, up to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6, before induction, a high amount of protein is obtained (relative to the amount of medium used), with isotopic labeling of approximately 97%. Despite this good result, there is a percentage of the \u003csup\u003e13\u003c/sup\u003eC-D-glucose that is used just to generate biomass and therefore it is \u0026ldquo;wasted\u0026rdquo; to improve the yield of labeled protein.\u003c/p\u003e\n\u003cp\u003eA simple way to improve this would be to combine this protocol with a first step of biomass generation in rich media, similar to other protocols, which then switch to labeled media by centrifugation and produce high yields of labeled proteins. One drawback of this approximation is that centrifugation steps can be stressful for the bacteria, and they recover slowly. In fact, the OD\u003csub\u003e600\u003c/sub\u003e can drop in the first few moments in minimal media, and it is difficult to estimate how long the bacteria need to be grown in these media before induction to maximize expression and minimize detrimental unproductive consumption of labeled nutrients.\u003c/p\u003e\n\u003cp\u003eTherefore, it can be hypothesized that the complete consumption of unlabeled glucose in minimal media could be as efficient in terms of biomass production as the use of rich media but avoid the stress of centrifugation. It has been reported that \u003cem\u003eE. coli\u003c/em\u003e recover quickly from short periods of starvation with no apparent sequelae (Lempp et al. 2019). In addition, the cells adapt to grow in these minimal media from the beginning, further reducing the stress of switching from rich to poor media and minimizing the time required to incorporate labeled metabolites.\u003c/p\u003e\n\u003cp\u003eTo evaluate this hypothesis, biomass production and glucose consumption were monitored under different conditions (Fig.\u0026nbsp;1). Different initial D-glucose concentrations were tested. In all the samples, after 23 h of growth at 25\u0026deg;C, 0.5% D-glucose was added, and the culture continued to grow at 30\u0026deg;C for 1.5 h, followed by growth at 20\u0026deg;C for another 24 h. The depletion of glucose after overnight growth was complete under all the conditions tested, and the growth rate recovery appeared to be complete after the addition of supplemental 0.5% D-glucose. Although it was predictable that some of this additional D-glucose would be consumed during the isotope integration step, after 1.5 h at 30\u0026deg;C, the remaining nutrient content was extremely low for the cultures with high initial glucose concentrations, leaving less than 0.1% D-glucose available for the protein expression step under the initial 0.5% D-glucose condition, and after 3 additional hours at 20\u0026deg;C, no glucose remained for 0.3, 0.4 or 0.5% initial glucose conditions.\u003c/p\u003e\n\u003cp\u003eThus, a counterintuitive result was found: it is not convenient to produce large amounts of biomass but rather to find a compromise between biomass and the consumption of labeled glucose to improve the final protein yield.\u003c/p\u003e\n\u003cp\u003eThe second variable monitored was the incorporation of \u003csup\u003e13\u003c/sup\u003eC into the labeled protein. The data show that for high initial glucose, only partial incorporation into the protein was reached, but when the initial glucose was reduced from 0.5\u0026ndash;0.4% and to 0.2%, the incorporation increased from 75\u0026ndash;90% and 98%, respectively, as detected by NMR (Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003eThese results led to the testing of other conditions - decreasing the glucose in the biomass generation step and increasing the labeled glucose added in the \u0026ldquo;isotope integration step\u0026rdquo; -. The protein yield under each new condition was greater for 0.2\u0026ndash;0.3% D-glucose in the \u0026ldquo;biomass generation step\u0026rdquo; combined with 1% D-glucose added in the \u0026ldquo;isotope integration step\u0026rdquo; (Fig. 3).\u003c/p\u003e\n\u003cp\u003eConsidering the data from the previous experiments, the effect of varying the time of the \u0026quot;isotope integration step\u0026quot; was tested by monitoring the protein yield and \u003csup\u003e13\u003c/sup\u003eC incorporation (Fig. 4). A second counterintuitive fact appeared: there is a minimal effect of the length of this phase on \u003csup\u003e13\u003c/sup\u003eC incorporation, which is always approximately 97\u0026ndash;98% and can even be eliminated.\u003c/p\u003e\n\u003cp\u003eAs the initial data indicated that for 0.5/0.5% conditions, even with a 1.5 h isotope integration step, the incorporation was approximately 75% it seems that in any case, the relative ratio between the initial unlabeled and subsequently added labeled glucose should not exceed 20% (0.2% initial D-glucose, 1% labeled D-glucose).\u003c/p\u003e\n\u003cp\u003eAlthough all biomass generation cultures were performed at 25\u0026deg;C to ensure that maximum O\u003csub\u003e2\u003c/sub\u003e was available to the cells, the influence of temperature on growth was monitored at 25\u0026deg;C, 30\u0026deg;C and 37\u0026deg;C (Fig. 5). Complete glucose depletion is reached in overnight cultures at 25\u0026deg;C or 30\u0026deg;C, whereas at 37\u0026deg;C, complete glucose depletion is achieved after approximately 5.5 hours. This would allow the biomass generation step to be shortened. However, the high growth rate at 37\u0026deg;C could have disadvantages, such as a lower number of ribosomes per cell (Marr 1991) or a possible microaerobic state, which could promote the accumulation of acetate (Partridge et al. 2007) and therefore inhibit cellular growth or the expression of the desired protein (Shiloach and Fass 2005). Finally, other \u003cem\u003eE. coli\u003c/em\u003e strains or plasmid‒strain combinations may be less efficient in nutrient consumption than those tested here and may require longer culture times. Although the total time for 25\u0026deg;C or 30\u0026deg;C cultures is longer, the active time for the present method decreases from approximately 1\u0026ndash;2 hours (due to measurements of the OD\u003csub\u003e600\u003c/sub\u003e until it reaches 0.6, centrifugation, resuspension in minimal medium, and addition of inductor) to approximately 5\u0026ndash;10 minutes (due to the addition of labeled nutrients and inductor simultaneously). Taking in consideration all these facts, to grow the culture at 25\u0026ordm;C is recommended for this method.\u003c/p\u003e\n\u003cp\u003eAlthough we have not tested the possibility of starting the culture directly from colonies from a plate instead of an overnight liquid preinoculum culture, as previously reported (Sivashanmugam et al. 2009), it could also diminish the total time of culture in addition to the already diminished active time.\u003c/p\u003e\n\u003cp\u003eThe influence of different glucose concentrations during the expression step on yield was also investigated (Fig. 6). A slight increase in yield was detected when the glucose concentration increased from 1\u0026ndash;1.5%, but the yield decreased at higher percentages. In any case, the increments are in the range of error, so there is no evident advantage in the use of higher glucose percentages. In any case, this method clearly improves the yield of labeled protein compared with some of the previously described protocols, and even in the least favorable case, there is a 20% increase. The tests indicate that 50 mL of culture (0.5 g of \u003csup\u003e13\u003c/sup\u003eC-D-glucose) is sufficient to obtain 10\u0026ndash;20 mg of purified labeled protein. Notably, Sivashanmugam\u0026apos;s protocol was modified, and 3 times more NH4Cl was used for this experiment. When the original amounts were used, the yield was drastically reduced (see Fig.\u0026nbsp;8 below), indicating the importance of an appropriate ratio of nutrients to obtain the maximum yield in any protocol.\u003c/p\u003e\n\u003cp\u003eThe ratios of NH4Cl and D-glucose in this recipe were calculated to ensure that glucose was the limiting nutrient. In this way, it can be ensured that all the unlabeled glucose has been depleted in the biomass production step. Since \u003csup\u003e13\u003c/sup\u003eC-labeled proteins for NMR are usually also \u003csup\u003e15\u003c/sup\u003eN-labeled, \u003csup\u003e15\u003c/sup\u003eNH4Cl must be used from the beginning because it cannot be assured that there is no remaining nitrogen that has not been consumed in the initial steps. Since \u003csup\u003e15\u003c/sup\u003eNH4Cl is much cheaper than \u003csup\u003e13\u003c/sup\u003eC-D-glucose, net savings still prevail, and it will be even more significant if we need to use D7-\u003csup\u003e13\u003c/sup\u003eC-D-glucose or other isotope-labeled precursors, which are even more expensive than \u003csup\u003e13\u003c/sup\u003eC-D-glucose.\u003c/p\u003e\n\u003cp\u003eFinally, two experiments were carried out to test whether this method could be used universally, regardless of the strain or protein expressed. To check for strain independence, CNRP1 was expressed in the BL21star(DE3), Turner\u0026trade;(DE3), C41(DE3), and Shuffle\u0026reg; T7 LysY strains, and \u003csup\u003e13\u003c/sup\u003eC incorporation was determined by mass spectrometry. No differences in glucose uptake or protein expression were found among these strains and the \u003csup\u003e13\u003c/sup\u003eC incorporation was almost identical (97.6%, 97.4%, 97.9% and 96.9%, respectively) (Fig.\u0026nbsp;7). To determine the variations of yield in the expression of different proteins depending of the protocol used, the method was tested for proteins CNRIP1, PHOX2b XS and NEX XF1 (Fig.\u0026nbsp;8). In all of them, a higher quantity of protein was obtained with the method described here than with the other protocols. Notably, the ratios between the different methods are not exactly the same for the 3 proteins, but in any case, the yield per gram of glucose is higher with this method for the three proteins tested.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eA new method for the production of \u003csup\u003e13\u003c/sup\u003eC-labeled proteins has been developed (a detailed protocol is provided in the supplemental material), which minimizes the time commitment required (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) and optimizes the consumption of labeled nutrients.\u003c/p\u003e \u003cp\u003eAn initial culture step at 25\u0026ndash;30\u0026deg;C in minimal medium allows biomass to be generated but avoids centrifugation. No \"isotope integration step\" is necessary, and simply adjusting the temperature to the appropriate level for protein expression before adding labeled nutrients and the inductor is enough to obtain maximum protein yields with high \u003csup\u003e13\u003c/sup\u003eC incorporation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis protocol, with minimal variations, could be used to generate other labels, such as selenomethionine for X-ray crystallography and for the selective labeling of different amino acid positions using 1,3-\u003csup\u003e13\u003c/sup\u003eC-glycerol or 2-\u003csup\u003e13\u003c/sup\u003eC-glycerol or other precursors as carbon sources. As well as for triple (\u003csup\u003e2\u003c/sup\u003eH, \u003csup\u003e15\u003c/sup\u003eN, \u003csup\u003e13\u003c/sup\u003eC) labeled proteins.\u003c/p\u003e \u003cp\u003eIn summary, two unexpected and counterintuitive findings, the limited biomass generation and the irrelevance of the isotope integration step, have allowed to develop a highly optimized protocol that offers significant advantages over other published protocols: simplicity, no need for monitoring, minimal active time, high yields of labeled protein and cost reduction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.A.T. to the study conception and design the study, made the experiments and wrote the manuscript.. Material preparation, data collection and analysis were performed by [full name], [full name] and [full name]. The first draft of the manuscript was written by [full name] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe author thanks Daniel Calvo for technical assistance, David Pantoja-Uceda for assistance with NMR spectroscopy, Pl\u0026aacute;cido Galindo from the mass spectrometry data adquisition, and Douglas V. Laurents for constructive feedback. Proteins were expressed, and NMR spectra were recorded at the Manuel Rico NMR Laboratory (LMR), a node of the ICTS for biomolecular NMR (R-LRB).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnt\u0026oacute;n R, Trevi\u0026ntilde;o M\u0026Aacute;, Pantoja-Uceda D, F\u0026eacute;lix S, Babu M, Cabrita EJ, Zweckstetter M, Tinnefeld P, Vera AM, Oroz J (2024) Alternative low-populated conformations prompt phase transitions in polyalanine repeat expansions. 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Protein Sci 18:936\u0026ndash;948. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pro.102\u003c/span\u003e\u003cspan address=\"10.1002/pro.102\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"journal-of-biomolecular-nmr","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnmr","sideBox":"Learn more about [Journal of Biomolecular NMR](http://link.springer.com/journal/10858)","snPcode":"10858","submissionUrl":"https://submission.nature.com/new-submission/10858/3","title":"Journal of Biomolecular NMR","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Protein expression, glucose consumption, minimal active time, cost reduction, 13C labeling","lastPublishedDoi":"10.21203/rs.3.rs-5123333/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5123333/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNMR is a powerful tool for the structural and dynamic study of proteins. One of the necessary conditions for the study of these proteins is their isotopic labeling with \u003csup\u003e13\u003c/sup\u003eC, \u003csup\u003e15\u003c/sup\u003eN and sometimes \u003csup\u003e2\u003c/sup\u003eH. One of the most widely used methods to obtain these labeled proteins is heterologous expression of the proteins in \u003cem\u003eE. coli\u003c/em\u003e using \u003csup\u003e13\u003c/sup\u003eC-D-glucose and \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl as the sole nutrient sources. In recent years, the price of \u003csup\u003e13\u003c/sup\u003eC-D-glucose has almost tripled, making it essential to develop labeling methods that are as cost effective as possible. In this work, different parameters were studied to achieve the most rational use of \u003csup\u003e13\u003c/sup\u003eC-D-glucose, and an optimized method was developed to obtain labeled proteins with high labeling and low \u003csup\u003e13\u003c/sup\u003eC-D-glucose consumption. Surprisingly, the optimized method is also simple and does not require monitoring of culture growth.\u003c/p\u003e","manuscriptTitle":"Counterintuitive method improves yields of isotopically labeled proteins expressed in flask-cultured Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-22 09:22:15","doi":"10.21203/rs.3.rs-5123333/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-02T14:06:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-27T21:13:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-21T10:04:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8820266953599065001390278550729429852","date":"2024-11-14T16:22:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218245030032479321142602773826494955975","date":"2024-11-04T08:45:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-21T15:02:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-14T18:20:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-21T05:33:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Biomolecular NMR","date":"2024-09-20T11:36:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-biomolecular-nmr","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnmr","sideBox":"Learn more about [Journal of Biomolecular NMR](http://link.springer.com/journal/10858)","snPcode":"10858","submissionUrl":"https://submission.nature.com/new-submission/10858/3","title":"Journal of Biomolecular NMR","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ac8310c2-48f2-45ed-be23-e2a80c145452","owner":[],"postedDate":"January 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-03T16:02:51+00:00","versionOfRecord":{"articleIdentity":"rs-5123333","link":"https://doi.org/10.1007/s10858-025-00461-2","journal":{"identity":"journal-of-biomolecular-nmr","isVorOnly":false,"title":"Journal of Biomolecular NMR"},"publishedOn":"2025-03-01 15:58:06","publishedOnDateReadable":"March 1st, 2025"},"versionCreatedAt":"2025-01-22 09:22:15","video":"","vorDoi":"10.1007/s10858-025-00461-2","vorDoiUrl":"https://doi.org/10.1007/s10858-025-00461-2","workflowStages":[]},"version":"v1","identity":"rs-5123333","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5123333","identity":"rs-5123333","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}