Engineering self-cleavage fusion system for the Production of Chimera Spider Silk Proteins

Engineering self-cleavage fusion system for the Production of Chimera Spider Silk Proteins | 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 Engineering self-cleavage fusion system for the Production of Chimera Spider Silk Proteins bixia zhou, Yufan Huang, Yongqin Su, Bingrui An, Mi Shen, Ke Zheng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3905454/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The spider silk protein (spidroin), which has powerful mechanical properties, has been extensively studied and shown potential application in various fields. The predatory nature of spiders makes native spidroin challenging to obtain, while heterologous expression of spidroin was hindered by the gene sequence features such as highly repetitive regions and high GC content. The low yield of spidroin subsequently affects its further application. In this study, we constructed a convenient expression system by employing a fusion tag in combination with a self-cleavage intronic peptide (intein) for three kinds of chimeric spidroins with different numbers of repetitive units, and soluble expression of the three kinds of spidroins after optimizing expression conditions was achieved with yields of 266 mg/L (NT2RepCT), 135 mg/L (NT4RepCT), and 125 mg/L (NT6RepCT), respectively. Three kinds of chimeric spidroins displayed increased β-sheet content with increased repetitive units during the transition from the solution to the dry state. Their capacity to form filamentous fibrils increased with the number of repetitive numbers. This study provides a solution for spidroin soluble expression and lays a foundation for its future application. chimeric spider silk protein fusion tag self-cleavage intein soluble expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Spiders use different glands to produce up to seven types of silk with different mechanical properties 1 , 2 . Among them, the major ampullate gland produces major ampullate silk, also known as dragline silk. The major ampullate silk commonly used to form the skeletal structure of spider webs 1 is the strongest of all known spider silks; it is three times stronger than aromatic nylon fibers and five times stronger than steel 3 . It is a potential biomaterial for military, industrial, and medical purposes owing to its superb biodegradability and biocompatibility 4 . The unique mechanical properties of spider silk stem from the unique amino acid sequence of the spidroin 5 . The primary structures of the spidroins are all highly similar, containing a highly conserved and highly conserved N-terminal nonrepeat region (NT), a C-terminal nonrepeat region (CT), and a central repeat region (Rep) at the core. The NT and CT are mainly responsible for maintaining stable storage of spidroin in the silk gland and controlling spider fiber formation 6 . The Rep accounts for more than 90% of the spidroin sequence and determines the materialistic properties of different spider silk fibers 7 – 9 . Mashelkar, R. A. et al. demonstrated that spidroins with a greater number of REP allow for more interchain and intrachain interactions and had fewer chain end defects, thus improving the mechanical properties of the spider silk fibers 10 . In addition, usually, REP is characterized by a high degree of repetition and a high content of specific amino acids. However, since large-scale spider breeding is unfeasible, genetic engineering techniques are the best option for obtaining spidroin. Genetic engineering is widely used to produce large amounts of spidroin because of the cannibalistic and territorial nature of spiders, and E. coli is the most commonly used host cell for heterologous expression. However, due to the lack of a post-translational modification system, various functional components suffer from poor adaptability, unstable structures, and issues, such as low yields, poor stability, and inclusion body formation, which are usually encountered when these substances are expressed in E. coli 11 , 12 . In particular, the large number of repetitive units and its high GC content in the spidroin gene make the heterologous expression more challenging, resulting in the difficulty and complexity in further bio-design and modification for practical use of spidroins. Current techniques for making artificial spider silk include denaturing conditions 13 , 14 and electrospinning 15 . The solubility of recombinant spidroin after being solubilized with solvents like hexafluoroisopropanol (HFIP) is still lower than the solubility of native (30–50% w/v) 16 ; what is more, the solubility of recombinant spidroin affects the mechanical properties of its subsequent spinning, and the mechanical capacity of artificial silk spun from low-solubility spidroins is not able to be comparable to that of the silk spun from the native, highly-solubilized spidrion comparable to those spun from native highly soluble spidroins 17 . Thus, developing a suitable method for highly soluble expression is urgently needed for mass production and better mechanical properties of artificial silk. Fusion tag technology is a standard tool for soluble expression of heterologous proteins. Thamm et al. illustrated that the yield of spidroin in E. coli was enhanced by utilizing a SUMO-tag 18 . Cedric Dicko et al. improved the yield of spidroin by using the green fluorescent protein (GFP), but soluble expression was not achieved 19 . However, introducing fusion tags for expression requires the introduction of exogenous proteases, such as enterokinase and TEV protease, during the isolation and purification of the target protein 20 , increasing the difficulty of subsequent purification. An intrinsic peptide (Intein) is a mobile genetic element that can be used to precisely isolate target proteins without adding exogenous proteases 21 , 22 . Mini-Intein ∆I-CM, an intronic peptide with self-cleaving activity at the C-terminal only, was developed by Wood et al. 23 . Then Sun et al. established a novel soluble protein expression system based on the synergistic interaction of elastin-like polypeptide (ELP) and Mini-Intein ∆I-CM, the fusion of which allowed the target protein to be directly separated from ELP in a mild and enzyme-free environment, allowing for more efficient isolation and purification of the target protein 24 . To obtain a highly soluble form of spidroin for subsequent spinning, we constructed a "fusion tag-intein-target protein" system using the fusion tag NusA in combination with the intrinsic peptide Mini-Intein ∆I-CM for the expression of three kinds of chimeric spidroins with different numbers of repetitive units. In addition, the repetitive units in spidroin are believed to benefit the formation of beta-sheets and nanofibrils 25 . Therefore, we further investigated the changes in the secondary structure of the three kinds of chimeric spidroins during fibril formation and self-assembly. The availability of soluble spidroin allows further high-performance applications to be easily achieved. The expression and purification system developed in our study can be applied to produce proteins that are difficult to express and simplify the pretreatment process. High-performance biomimetic spidroin materials with practical applications are expected to be obtained. This study also provides theoretical references for preparing polymer materials with other complex mesoscopic structural features, such as collagen and amyloid protein. 2. Materials and Methods 2.1. Construction of the NusA-Intein-NTnRepCT expression system The bacterial strains, plasmids, the sources of the gene sequences, and primers used in this study are listed in Additional File 1, Table S1 . The pET43.1a (+)-NusA-Intein-NT2RepCT expression vector used in this work was created by combining the target gene fragment (NT2RepCT) with the pET43.1a (+)-NusA-Intein vector via a double digestion-ligation (HindIII, XhoI, TAKARA) process (produced by Genewiz). (NTR2CT, NT: NCBI Accession number. AM259067; CT: NCBI Accession number JX513956; R2: NCBI Accession number. AJ973155) Overall sequence analysis was performed using ProtParam (ExPASy), and hydrophobicity analysis was performed via ProtScale. ( https://web.ExPASy.org/protscale/ ) The subsequent polyploid expression vector pET43.1a-NusA-Intein-NTnRepCT was generated via a head and tail strategy 26 , using primers F2 and R2 (NheI and SpeI) with two compatible but non-reproducible restriction endonuclease sites (Additional File 1, Table S1 ). NT2RepCT was used as a template to amplify the repeat module gene (R2), which was double digested (NheI and SpeI, TAKARA) and subsequently ligated to the single digested and expression vector pET43.1a-NusA-Intein-NT2RepCT. This process was repeated several times to obtain the fusion protein expression vector pET43.1a-NusA-Intein-NT2Rep-6RepCT with different numbers of repetitive units. A diagram of the construction process is shown in Fig. 1 . To verify the role of the fusion tag NusA in enhancing the soluble expression of spidroins, three expression vectors pET43.1a-NT2Rep-6RepCT without the fusion tag NusA and intein served as a parallel control group, and the resulting recombinant plasmids were transformed into E. coli BL21(DE3) competent cells for transformation and a single colony was selected for PCR identification and sequenced at Genewiz (Suzhou, China). 2.2. Optimization of protein expression conditions E. coli BL21 (DE3) cells carrying the expression plasmid (available from section 2.2) were induced with IPTG (0.1 mM) for 12 h at 30°C. After induction and shattering treatments, protein expression was verified by SDS-PAGE. Subsequently, the effects of inducer concentration, induction temperature, and induction time on recombinant protein expression were considered, and the specific conditions were optimized to determine the optimal conditions for protein expression. Different temperatures, such as 16°C, 20°C, 25°C, 30°C, and 35°C, and different concentrations of the induction agent, 0.1 mM, 0.3 mM, 0.5 mM, and 1.0 mM, were used to induce the expression of the recombinant construct and empty vector in BL21(DE3) transformants for 6 h, 12 h, 24 h, 36 h, and 48 h, respectively. Finally, the optimal expression conditions were determined by soluble proportion and yield via SDS-PAGE. Total protein and soluble fractions were calculated by grey-scale scanning using Quantity One version 4.62 software (Bio-Rad, USA). Protein concentrations were determined using a BCA kit from Vazyme (Nanjing, China). 2.3. Protein purification The His-tagged recombinant proteins NT2Rep-6RepCT were purified using Ni 2+ -NTA agarose (QIAGEN) 27 . The Ni2+-NTA agarose column was equilibrated with a binding buffer containing 20 mM Tris-HC l and 140 mM NaCl (pH 8.0) and eluted with an elution buffer containing 20 mM Tris-HCl, 300 mM NaCl, and 0.5 M imidazole (pH 8.0). A centrifugal ultrafiltration device (Vivaspin 20, GE Healthcare) with a molecular weight of 10 kDa was used to concentrate the spidroin and validate the analysis by SDS‒PAGE. 2.4. Protein morphology observation The purified and freeze-dried spidroin was broken by freezing in liquid nitrogen and then sputter-coated with gold on the surfaces of the proteins. The surface and cross-section of the prepared spidroins were observed under a scanning electron microscope (FESEM, Hitachi S4800, Japan). 2.5. Secondary structure analysis Circular Dichroism (CD): Recombinant spidroin conformations were determined by circular dichroism (JASCO J-1500 CD Spectrometer) in the far ultraviolet region in the wavelength range 185–260 nm, and a known concentration of chimeric protein stock solution was obtained and diluted to a concentration of 0.5 mg/L. Fourier Transformed Infrared Spectroscopy (FTIR) (Bruker OPTIK GmbH Tensor II, Germany) was used to analyze the secondary structure and content of the components in the chimeric spider silk fibrils, which were dried and used for spectroscopic determination using an attenuated total reflection detector with a scanning range of 4000 − 400 cm − 1 and a resolution of 8 cm − 1 and 128 scans. The spectra were processed by the software Peakfit V 4.2, and the Raman spectra in the amide I band (1700 − 1600 cm − 1 ) were fitted with second-order derivatives to obtain the content of each component by calculating the peak area. 2.6. Self-assembly ability analysis The morphology of the chimeric arachnoid nanofibers was observed using atomic force microscopy (Bruker ICON). Before testing, the chimeric spidroin stock solution was brought to a concentration of 0.001% (w/v), and the diluted solution was subsequently dried dropwise on the surface of the mica flakes. The morphology was measured using the scanning mode and a 2 nm radius probe with a mechanical modulus of 0.4 N/m. The final nanofiber morphology and mechanical data were analyzed using NanoScope Analysis 1.8, a software program that comes with the instrument. 3. Results 3.1. Expression of NTnRepCT spidroin (n=2, 4, and 6) and their purification The spidroin gene sequence contains a highly conserved N-terminal nonrepeat structural domain (N-terminal, NT), a C-terminal nonrepeat region (CT), and a core repetitive unit (REP). This study evaluated the spinning performance of the recombinant spidroins (NT2Rep-6RepCT) with different amonts of repetitive units. The recombinant spidroins constructed in this study were selected from the Rep of Euprosthenops australis MaSp1, which is characterized by high amino acid levels, with Ala (A) and Gly (G) accounting for 67.8% of the total amino acids. However, this gives the nucleotide sequence a high GC content of 86%. These amino acids are primarily GGX, GX (X for other amino acids), and poly-Ala, typical of the spidroin module. The high repetitiveness of spidroin sequence poses a great challenge for its heterologous production, which results in the formation of truncated proteins, low expression, and inclusion bodies. Therefore, we constructed a "fusion tag-intein-spidroin" expression system to achieve soluble expression of spidroin. The hydrophilicity/hydrophobicity changed periodically with increasing core repetitive units and was uniformly distributed. (Additional File 1: Figure. S1 ) This amphiphilicity may also correlate with the disorganization of the spidroins, and an increase in disorganization may improve the ability of the spidroins to self-assemble. The self-assembly properties of soluble spidroins are related to the composition of their hydrophilic and hydrophobic structural domains 28 . Among these domains, hydrophobic structural domains are the key element in the phase separation of spidroins, which is the early stage of protein self-assembly 29 . The hydrophobic structural domains of spidroins are largely disordered, and the self-association of hydrophobic sequences provides the driving force for phase separation 30 . The greater the disorder of the protein sequence is, the stronger the intrinsic structural stability of its phase separation state, and subsequently the stronger the self-assembly ability of its protein. To obtain soluble expression of spidroin in E. coli , a recombinant expression system of the fusion tag-intein-spidroin was constructed by introducing the fusion tag NusA and the intrinsic peptide Intein with self-cleaving function. A parallel control group without the fusion tag was constructed to verify the effect of the fusion tag NusA in increasing the solubility of spidroins. For subsequent purification, a histidine tag (6×His tag) was added to the gene end (shown in Figure 1 ). The final fusion protein containing different numbers of repetitive units in the core region of the spidroin "NusA-Intein-NTnRepCT" was expressed in E. coli . The expression of the three chimeric spidroins is shown in Figure 2 . All three spidroins in the control group were expressed as inclusion bodies in very few amounts, and barely any bands appeared in the soluble fraction (Figure 2a) . Their theoretical molecular weights are approximately 32, 40, and 46 kDa, respectively. The apparent molecular weights of the fusion proteins NusA-Intein-NT2RepCT and NusA-Intein-NT4RepCT in the soluble fractions by SDS-PAGE are generally consistent with the theoretical molecular weights. In contrast, the apparent molecular weights are higher for several repetitive units of 6 (6Rep). This may be because spidroin is a special kind of self-assembling protein, which results in a high apparent molecular weight (56 kDa) due to its structure affecting its ability to migrate in SDS-PAGE. However, by employing the fusion tag NusA, a small amount of expression of chimeric spidroins appeared in the soluble fraction after the self-cleavage by intein. To achieve higher expression levels, the expression conditions for the three spidroins NT2Rep-6RepCT were optimized at 0.3 mM IPTG and induced for 24 h at 20°C (Figure 2b) . On the one hand, these findings suggested that the fusion tag NusA contributes to the soluble expression of spidroin. On the other hand, for self-cleavage, a microacidic environment will be created in the E. coli expression system as the secondary products gradually accumulate. In this environment, the target protein (NT2Rep-6RepCT) is produced after self-cleavage by intein to remove NusA-Intein 31 . Specifically, due to the large molecular weight of NT6RepCT, self-cleavage of the NusA tag and intein may not be sufficient resulting in a large NusA-intein portion in the soluble fraction. ( Figure 2b ) Subsequently, three kinds of chimeric spidroins were purified through Ni-chelating affinity chromatography. All three chimeric proteins reached a purity of over 95 % by grey-scale scanning. 266 mg/L (NT2RepCT), 135 mg/L (NT4RepCT), and 125 mg/L (NT6RepCT) were determined by the BCA method for the three purified chimeric proteins ( Figure 3) . The yields were significantly higher compared to those reported in other literature (40 mg/L 32 , 125 mg/L 18 ), thus indicating that the fusion tag NusA effectively increased the soluble expression of the three chimeric spidroins in E. coli . As shown in Figure 4 , a clear strong positive peak at around 190-195 nm and negative peaks at around 208 and 222 nm indicated that the secondary structure of three kinds of chimeric spidroins are mainly α-helix and random coil structures. This result was in good agreement with that of completely dissolved proteins (for example, recombinant spidroin 33 and regenerated silk protein 34 ), suggesting a good dissolvability of these three chimeric proteins in aqueous solutions. In particular, the helical structure in these three chimeric proteins was critical to their self-assembling into ordered formations. After that, the self-assembly of chimeric spidroin was investigated by AFM observation. As shown in Figure 5 .a.c.e , the three kinds of spidroin s were initially folded into nanoparticles with an average 4-6 nm diameter. The self-assembly of silk proteins generally followed a nucleation process, where nanoparticles provided heterogeneous sites to introduce fibrillar formation in solutions. However, the NT2RepCT could barely form nanofibrils after incubating at 60 °C for 48 h ( Figure 5b ), probably due to the protein's lower molecular weight (only 2 of the repetitive units in the core region). The initial nanoparticles of NT2RepCT were slightly aggregated into large ones with a diameter of 10-25 nm at this condition. For comparison, the NT4RepCT and NT6RepCT with higher molecular weights presented fascinating network-like nanofibril structures at the same incubation condition (60 °C for 48 h), shown in Figure 5 d and 5f . The diameters of these nanofibrils were approximately 6 nm, which was consistent with the diameters of their preliminary nanoparticles ( Figure.5c and 5e ). In addition, the FTIR spectra confirmed the presence of ordered structure, i.e., β-sheet conformation in the self-assembled proteins. As shown in Additional File 1, Figure. S2 , the absorption peak at 1620 cm -1 is assigned to the β-turn conformation, while the peaks at 1646 and 1675 cm -1 are assigned to the random coil/helix and β-turn conformation, respectively. As shown in Additional File 1, Figure. S3, the deconvolution results of the FTIR spectra (amide I band) suggested an increase in β-sheet content, followed by an increasing number of repetitive units in the core region of proteins. The highest β-sheet content of 41.3% was presented in the NT6RepCT nanofibrils, compared to 39.1 and 38.3% in NT2RepCT and NT4RepCT samples, respectively (Additional File 1, Table S2 ). This is expected because the highly repetitive domains in protein sequences are believed to contribute to the transformation of β-sheets in recombinant spidroins. Notably, the self-assembly of NT4RepCT and NT6RepCT was very similar to that of native silk proteins as was the case for recombinant spidroins in other works 35,36 , as indicated by both the secondary structures and morphology of these nanofibrils. This self-assembly of these two proteins is promising for generating macroscale materials with robust mechanical properties. 3.2. Structural characterization of NTnRepCT The three chimeric spidroins exhibited different aggregation morphologies after freeze-drying under the same conditions. Figure 6 shows scanning electron microscopy (SEM) images of the resulting three spidroins, in which sample 2Rep could not form a large aggregation state due to the lack of repetitive units. This might result from the relatively small size of the repetitive core region of 2Rep spidroins (~32 kDa), which was unfavorable for initializing any fibrillar formation. However, 4Rep and 6Rep formed relatively dense interlaced and reticular structures, the surface of 4Rep spidroins had severe holes, and the aggregation of 6Rep spidroins was denser and more easily distributed. 4. Discussion and Conclusion Key findings are as follows: The three chimeric spidroins in this study consisted of repetitive sequences with high alanine and glycine contents, which resulted in low recombinant expression and a high tendency to form inclusion bodies. while natural spiders produce soluble spidroins in their glands. The solubility of recombinant spidroins plays a significant role in the mechanical performance of artificial fibres after subsequent spinning 17 . Previously, Bowen 12 and Xia et al. 11 selected repeated key spidroin sequences from the natural spidroin MaSp for heterologous expression and obtained spidroins of different molecular weights expressed as inclusion bodies, which indicated the significant challenge of producing soluble recombinant spidroins that contain highly repetitive sequences, and the need to find an effective strategy for achieving soluble expression of spidroins. Here we compare the proposed strategy with conventional methods 14,37 , which are different from those reported previously. The fusion tag NusA, together with the self-cleavage functional Intein, effectively reduces the formation of inclusion bodies and results in the soluble expression of spidroins. By employing a self-cleavage fusion system, the highest yield of NT2RepCT reached 266 mg/L in the study. Moreover, soluble helices facilitate subsequent spinning, and artificial silk spun from soluble spidroin may have superior mechanical properties. This established system enhanced the heterologous expression of spidroins, providing a reference for the soluble expression of difficult-to-express foreign proteins. The secondary structure compositions of the three kinds of chimeric spidroins with different morphologies were observed by CD and FTIR, and the trend of secondary structure transformation during morphological changes was observed. In an aqueous solution, NT2Rep-6RepCT existed mainly in the α-helix conformation, but in the solid state, the β-sheet content was 38.3%-41.3%. By comparing the secondary structure compositions of the chimeric spider silks in aqueous solution and in the solid state, we found that the three chimeric spider silks transformed from an α-helical to a β-sheet conformation during the drying process. A better morphology of chimeric spidroins with more repetitive units was observed by atomic force microscopy, which is consistent with the analysis of disorganization 38 . We consequently examined the self-assembly trends and capabilities of these materials after heat induction. The NT4RepCT and NT6RepCT, which have higher molecular weights, presented better network-like nanofibril structures, which indicated greater self-assembly capacity. These findings support the use of localized β-sheet structural domains in amorphous networks by Chan et al. to increase the mechanical toughness and stability. The resulting continuous β-sheet nanocrystal network obtained through grafting-from polymerization exhibited greater compressive strength and stiffness than the initial network lacking β-sheets 39 . In this study, we successfully expressed the three spidroins in a soluble form and confirmed their ability to self-assemble and form fibers. However, additional verification of their spinning performance is necessary. We acknowledge that there may be significant debate among researchers regarding protein yield and spinning performance. Although Xia et al. metabolically engineered the expression host in order to modify the production of spidroins and achieved a yield of 1.2 g/L by high-density fermentation, the expression of high-molecular-weight polyploid spidroins was still accompanied by putative truncated forms of the target protein, which prevented the complete preparation of high-molecular-weight spidroins 11 . Later, Qian et al. used Corynebacterium glutamicum as the host to establish a secretion production platform, and achieved a yield of 2.2 g/L by high-density fermentation. However, this method is limited to producing lower-molecular-weight spidroins (~40 kDa) 40 . Efficient secretion of high-molecular-weight spidroins via this method is challenging. Because our expression strategy proposed in this paper can provide another alternative method for the expression of spidroins, further high-density fermentation to obtain high yields of soluble spidroins and subsequent spinning validation are still l needed. In addition, our self-cleavage fusion system may serve as a reference for preparing high-performance spider silk materials for medical, military, and other fields. This approach might also lay a good foundation for the soluble expression of other proteins similar to spidroins, such as collagen and mussel proteins, and provide a theoretical reference for preparing other polymer materials with complex structures. Declarations Consent for publication: All the authors have been involved with the work and approved the manuscript for publication. Availability of data and materials: All data generated or analyzed during this study are included in this published article [and its supplementary information files] Conflict of Interest: The authors declare that the research was performed in the absence of any commercial or financial relationships that can be considered as a potential conflict of interest. Author Contributions: BXZ: Writing – original draft, Methodology. YFH: Investigation, Visualization. YQS: Conceptualization. BRA: Validation. MS: Data curation. KZ: Software, Resources. CC: Project Administration, Review & Editing. BFH: Funding acquisition, Supervision. Funding: This work was supported by scientific research plan projects of Shaanxi education department (22JC010), and Key R&D general projects of Shaanxi provincial department of science and technology (2023-YBNY-170), Natural science foundation of China (81973531), and the fundamental research project of the Shenzhen science and technology innovation commission (20200812211704001). References Lewis, R. V. Spider Silk: Ancient Ideas for New Biomaterials. Chem Rev 2006 , 106 (9), 3762–3774. https://doi.org/10.1021/cr010194g. Tokareva, O.; Jacobsen, M.; Buehler, M.; Wong, J.; Kaplan, D. L. Structure-Function-Property-Design Interplay in Biopolymers: Spider Silk. Acta Biomater 2014 , 10 (4), 1612–1626. https://doi.org/10.1016/j.actbio.2013.08.020. Blackledge, T. A.; Summers, A. P.; Hayashi, C. Y. Gumfooted Lines in Black Widow Cobwebs and the Mechanical Properties of Spider Capture Silk. Zoology (Jena) 2005 , 108 (1), 41–46. https://doi.org/10.1016/j.zool.2004.11.001. 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S.; Morton, Z.; Merwin, S.; Topilina, N. I.; Belfort, M. Intein Clustering Suggests Functional Importance in Different Domains of Life. Mol Biol Evol 2016 , 33 (3), 783–799. https://doi.org/10.1093/molbev/msv271. Novikova, O.; Topilina, N.; Belfort, M. Enigmatic Distribution, Evolution, and Function of Inteins. J Biol Chem 2014 , 289 (21), 14490–14497. https://doi.org/10.1074/jbc.R114.548255. Wood, D. W.; Wu, W.; Belfort, G.; Derbyshire, V.; Belfort, M. A Genetic System Yields Self-Cleaving Inteins for Bioseparations. Nat Biotechnol 1999 , 17 (9), 889–892. https://doi.org/10.1038/12879. Rai, K.; Chu, X.; Zhou, D.; Li, F.; Yang, J.; Lin, J.; Shen, S.; Song, H.; Yue, S.; Nian, R. Development of a Protein-Solubilizing Expression Method Based on the Synergistic Action of Intein ΔI-CM and the Solubility Enhancer Elastin-like Polypeptide. Biochemical Engineering Journal 2021 , 167 , 107900. https://doi.org/10.1016/j.bej.2020.107900. Hayashi, C. Y.; Shipley, N. H.; Lewis, R. V. Hypotheses That Correlate the Sequence, Structure, and Mechanical Properties of Spider Silk Proteins. International Journal of Biological Macromolecules 1999 , 24 (2–3), 271–275. https://doi.org/10.1016/S0141-8130(98)00089-0. O’Brien, J. P.; Hoess, R. H.; Gardner, K. H.; Lock, R. L.; Wasserman, Z. R.; Weber, P. C.; Salemme, F. R. Design, Synthesis, and Fabrication of a Novel Self-Assembling Fibrillar Protein ; 1994. Ueda, E. K. M.; Gout, P. W. B.; Morganti, L. Ni(II)-Based Immobilized Metal Ion Affinity Chromatography of Recombinant Human Prolactin from Periplasmic Escherichia Coli Extracts. Journal of Chromatography A 2001 , 922 (1–2), 165–175. https://doi.org/10.1016/s0021-9673(01)00875-5. Muiznieks, L. D.; Keeley, F. W. Phase Separation and Mechanical Properties of an Elastomeric Biomaterial from Spider Wrapping Silk and Elastin Block Copolymers. Biopolymers 2016 , 105 (10), 693–703. https://doi.org/10.1002/bip.22888. Borkner, C. B.; Lentz, S.; Müller, M.; Fery, A.; Scheibel, T. Ultrathin Spider Silk Films: Insights into Spider Silk Assembly on Surfaces. ACS Appl. Polym. Mater. 2019 , 1 (12), 3366–3374. https://doi.org/10.1021/acsapm.9b00792. Rauscher, S.; Pomès, R. Structural Disorder and Protein Elasticity. Adv Exp Med Biol 2012 , 725 , 159–183. https://doi.org/10.1007/978-1-4614-0659-4_10. Mills, K. V.; Johnson, M. A.; Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J Biol Chem 2014 , 289 (21), 14498–14505. https://doi.org/10.1074/jbc.R113.540310. Lin, Z.; Deng, Q.; Liu, X.-Y.; Yang, D. Engineered Large Spider Eggcase Silk Protein for Strong Artificial Fibers. Adv Mater 2013 , 25 (8), 1216–1220. https://doi.org/10.1002/adma.201204357. Shanafelt, M.; Rabara, T.; MacArt, D.; Williams, C.; Hekman, R.; Joo, H.; Tsai, J.; Vierra, C. Structural Characterization of Black Widow Spider Dragline Silk Proteins CRP1 and CRP4. MOLECULES 2020 , 25 (14). https://doi.org/10.3390/molecules25143212. Xu, S.; Li, X.; Zhou, Y.; Lin, Y.; Meng, Q. Structural Characterization and Mechanical Properties of Chimeric Masp1/Flag Minispidroins. Biochimie 2020 , 168 , 251–258. https://doi.org/10.1016/j.biochi.2019.11.014. Humenik, M.; Magdeburg, M.; Scheibel, T. Influence of Repeat Numbers on Self-Assembly Rates of Repetitive Recombinant Spider Silk Proteins. J Struct Biol 2014 , 186 (3), 431–437. https://doi.org/10.1016/j.jsb.2014.03.010. Tokareva, O. S.; Lin, S.; Jacobsen, M. M.; Huang, W.; Rizzo, D.; Li, D.; Simon, M.; Staii, C.; Cebe, P.; Wong, J. Y.; Buehler, M. J.; Kaplan, D. L. Effect of Sequence Features on Assembly of Spider Silk Block Copolymers. J Struct Biol 2014 , 186 (3), 412–419. https://doi.org/10.1016/j.jsb.2014.03.004. Metabolic engineering for recombinant major ampullate spidroin 2 (MaSp2) synthesis in Escherichia coli | Scientific Reports . https://www.nature.com/articles/s41598-017-11845-2 (accessed 2024-01-16). Sintya, E.; Alam, P. Self-Assembled Semi-Crystallinity at Parallel β -Sheet Nanocrystal Interfaces in Clustered MaSp1 (Spider Silk) Proteins. Materials Science and Engineering: C 2016 , 58 , 366–371. https://doi.org/10.1016/j.msec.2015.08.038. Chan, N. J.-A.; Gu, D.; Tan, S.; Fu, Q.; Pattison, T. G.; O’Connor, A. J.; Qiao, G. G. Spider-Silk Inspired Polymeric Networks by Harnessing the Mechanical Potential of β-Sheets through Network Guided Assembly. Nat Commun 2020 , 11 (1), 1630. https://doi.org/10.1038/s41467-020-15312-x. Jin, Q.; Pan, F.; Hu, C.-F.; Lee, S. Y.; Xia, X.-X.; Qian, Z.-G. Secretory Production of Spider Silk Proteins in Metabolically Engineered Corynebacterium Glutamicum for Spinning into Tough Fibers. Metab Eng 2022 , 70 , 102–114. https://doi.org/10.1016/j.ymben.2022.01.009. Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1.docx Supporting Information: Additional File 1. Strains, plasmids, and primers used in this study; Table S1, Proportion of secondary structure of chimera spider silk protein in solid state; Table S2, The hydropathicity/hydrophobicity of repetitive units; Figure. S1, Fourier Transform infrared spectra of chimeric NT2RepCT (black line) 、NT4RepCT (red line) and NT6RepCT (blue line); Figure. S2, Fourier Transform infrared spectra (amide I band) and fitted spectra of chimeric NT2RepCT(A)、NT4RepCT(B) and NT6RepCT(C); Figure. S3. Cite Share Download PDF Status: Posted Version 1 posted 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-3905454","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":270957089,"identity":"d9402a6f-8c49-44e5-8efa-5d528ba4fd48","order_by":0,"name":"bixia zhou","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"bixia","middleName":"","lastName":"zhou","suffix":""},{"id":270957090,"identity":"62165c0b-6944-453b-870f-4995a91f88bb","order_by":1,"name":"Yufan Huang","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Yufan","middleName":"","lastName":"Huang","suffix":""},{"id":270957091,"identity":"34d11e9e-1690-4df3-ba7b-c461f8401d6f","order_by":2,"name":"Yongqin Su","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Yongqin","middleName":"","lastName":"Su","suffix":""},{"id":270957092,"identity":"c35c6615-1f7a-428d-8af9-c8e548f27f20","order_by":3,"name":"Bingrui An","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Bingrui","middleName":"","lastName":"An","suffix":""},{"id":270957093,"identity":"43cf8576-9a88-40dd-910c-2ecbf9c0a3a0","order_by":4,"name":"Mi Shen","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Mi","middleName":"","lastName":"Shen","suffix":""},{"id":270957094,"identity":"51dc159b-f5d0-4261-966d-072320bd80ed","order_by":5,"name":"Ke Zheng","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Zheng","suffix":""},{"id":270957095,"identity":"1b87b41c-0ce6-4ee0-87ac-50824c4a81cf","order_by":6,"name":"Cheng Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACCRBRYcPA2MDAwAxkgmhitJxJY2BsI0kLY9thBgY2YrXwz24+9vAL2/k85vnNBz8XMNjIbjjA/OwBXkvuHEs3luG5XczYxpYsPYMhzXjDATZzA3xaDCRyzKQlJG4nNrbxmDHzMBxO3HCAh00Cv5b8b9ISBueAWvi/AbX8J0ZLDpvkh4QDIFvYgFoOENYicSPNTJrhQDJQS5qxNI9BsvHMw2xmeLXwz0h+Jvnzn13ixubDDz/zVNjJ9h1vfoZXCwgA3cPAYNgAdicDJHYIAcYfQEKeCIWjYBSMglEwQgEApENFcK6WLJEAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":true,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Cheng","suffix":""},{"id":270957096,"identity":"7c333c8d-ce10-4968-a5da-cd03412d2609","order_by":7,"name":"Bingfang He","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Bingfang","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2024-01-28 10:29:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3905454/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3905454/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50744069,"identity":"0639662f-c45b-404c-8c75-bdd3487b9814","added_by":"auto","created_at":"2024-02-06 16:38:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78172,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the construction of a multi-repeat module and recombinant spidroin expression vector construction.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3905454/v1/01103b2b02ef5fd84b5ff2ee.jpg"},{"id":50744067,"identity":"d87b469c-128a-441d-9a7f-062bd135be1c","added_by":"auto","created_at":"2024-02-06 16:38:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a). Tricine-SDS-PAGE analysis of three spidroins without the fusion tag NusA and intein. Note: M: protein marker; 1: control (empty vector); S: cell lysis of spidroin; IB: inclusion body\u003c/strong\u003e.\u003cstrong\u003e Arrows indicate the location of migration of the three spidroins. (b). Tricine-SDS‒PAGE analysis of the results obtained under conventional induction conditions and after optimization of the induction conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: “*”: use of optimized induction conditions; “*” indicates the use of conventional induction conditions.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3905454/v1/260fa38a510e3449c06e1c21.jpg"},{"id":50743562,"identity":"b2c58613-67ef-4aa3-b71c-25a9e3e96e71","added_by":"auto","created_at":"2024-02-06 16:30:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePurification of\u003c/strong\u003e \u003cstrong\u003eNT2RepCT, NT4RepCT and NT6RepCT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: protein marker (M); control (empty vector)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3905454/v1/61eeca6880ff48d9d987a818.jpg"},{"id":50744068,"identity":"26671c7f-6a5d-4487-ba2a-b5fbb22da769","added_by":"auto","created_at":"2024-02-06 16:38:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD spectra of NT2RepCT (Purple)、NT4RepCT (Orange) and NT6RepCT (Pink) in aqueous solution.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3905454/v1/7915767eea3b4f5117bb328a.jpg"},{"id":50743567,"identity":"d2e1e32e-4b4b-4818-8bb9-ec97f0e6e295","added_by":"auto","created_at":"2024-02-06 16:30:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":116337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAtomic force microscopy results for NT2RepCT (a), NT4RepCT (c) and NT6RepCT (e).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3905454/v1/448813aaa7a6c309d832ceab.jpg"},{"id":50743564,"identity":"21a3337f-64d7-4e76-9941-3f7e3a354718","added_by":"auto","created_at":"2024-02-06 16:30:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":123775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of NT2RepCT, NT4RepCT and NT6RepCT.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3905454/v1/b18de3c7a059917b3168cfd3.jpg"},{"id":52751204,"identity":"bc73f9f5-b40d-4c69-b1a6-8b54b4c5f5c3","added_by":"auto","created_at":"2024-03-15 10:24:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":860400,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3905454/v1/f9eaca1d-9d6b-4f31-b68c-2c4c9f3d5bd0.pdf"},{"id":50743568,"identity":"c77b9c93-df18-4e55-9b17-eeb1fa653308","added_by":"auto","created_at":"2024-02-06 16:30:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":278576,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupporting Information:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional File 1.\u003c/p\u003e\n\u003cp\u003eStrains, plasmids, and primers used in this study; Table S1, Proportion of secondary structure of chimera spider silk protein in solid state; Table S2, The hydropathicity/hydrophobicity of repetitive units; Figure. S1, Fourier Transform infrared spectra of chimeric NT2RepCT (black line) 、NT4RepCT (red line) and NT6RepCT (blue line); Figure. S2, Fourier Transform infrared spectra (amide I band) and fitted spectra of chimeric NT2RepCT(A)、NT4RepCT(B) and NT6RepCT(C); Figure. S3.\u003c/p\u003e","description":"","filename":"AdditionalFile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3905454/v1/2809e4291cd31df1228d2c0f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Engineering self-cleavage fusion system for the Production of Chimera Spider Silk Proteins","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpiders use different glands to produce up to seven types of silk with different mechanical properties\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Among them, the major ampullate gland produces major ampullate silk, also known as dragline silk. The major ampullate silk commonly used to form the skeletal structure of spider webs \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e is the strongest of all known spider silks; it is three times stronger than aromatic nylon fibers and five times stronger than steel\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It is a potential biomaterial for military, industrial, and medical purposes owing to its superb biodegradability and biocompatibility\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The unique mechanical properties of spider silk stem from the unique amino acid sequence of the spidroin\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The primary structures of the spidroins are all highly similar, containing a highly conserved and highly conserved N-terminal nonrepeat region (NT), a C-terminal nonrepeat region (CT), and a central repeat region (Rep) at the core. The NT and CT are mainly responsible for maintaining stable storage of spidroin in the silk gland and controlling spider fiber formation \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The Rep accounts for more than 90% of the spidroin sequence and determines the materialistic properties of different spider silk fibers\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Mashelkar, R. A. et al. demonstrated that spidroins with a greater number of REP allow for more interchain and intrachain interactions and had fewer chain end defects, thus improving the mechanical properties of the spider silk fibers \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In addition, usually, REP is characterized by a high degree of repetition and a high content of specific amino acids. However, since large-scale spider breeding is unfeasible, genetic engineering techniques are the best option for obtaining spidroin.\u003c/p\u003e \u003cp\u003eGenetic engineering is widely used to produce large amounts of spidroin because of the cannibalistic and territorial nature of spiders, and \u003cem\u003eE. coli\u003c/em\u003e is the most commonly used host cell for heterologous expression. However, due to the lack of a post-translational modification system, various functional components suffer from poor adaptability, unstable structures, and issues, such as low yields, poor stability, and inclusion body formation, which are usually encountered when these substances are expressed in \u003cem\u003eE. coli\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In particular, the large number of repetitive units and its high GC content in the spidroin gene make the heterologous expression more challenging, resulting in the difficulty and complexity in further bio-design and modification for practical use of spidroins. Current techniques for making artificial spider silk include denaturing conditions \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eand electrospinning\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The solubility of recombinant spidroin after being solubilized with solvents like hexafluoroisopropanol (HFIP) is still lower than the solubility of native (30\u0026ndash;50% w/v)\u003csup\u003e16\u003c/sup\u003e; what is more, the solubility of recombinant spidroin affects the mechanical properties of its subsequent spinning, and the mechanical capacity of artificial silk spun from low-solubility spidroins is not able to be comparable to that of the silk spun from the native, highly-solubilized spidrion comparable to those spun from native highly soluble spidroins\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Thus, developing a suitable method for highly soluble expression is urgently needed for mass production and better mechanical properties of artificial silk.\u003c/p\u003e \u003cp\u003eFusion tag technology is a standard tool for soluble expression of heterologous proteins. Thamm et al. illustrated that the yield of spidroin in \u003cem\u003eE. coli\u003c/em\u003e was enhanced by utilizing a SUMO-tag\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Cedric Dicko et al. improved the yield of spidroin by using the green fluorescent protein (GFP), but soluble expression was not achieved \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, introducing fusion tags for expression requires the introduction of exogenous proteases, such as enterokinase and TEV protease, during the isolation and purification of the target protein \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, increasing the difficulty of subsequent purification. An intrinsic peptide (Intein) is a mobile genetic element that can be used to precisely isolate target proteins without adding exogenous proteases \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Mini-Intein ∆I-CM, an intronic peptide with self-cleaving activity at the C-terminal only, was developed by Wood et al.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Then Sun et al. established a novel soluble protein expression system based on the synergistic interaction of elastin-like polypeptide (ELP) and Mini-Intein ∆I-CM, the fusion of which allowed the target protein to be directly separated from ELP in a mild and enzyme-free environment, allowing for more efficient isolation and purification of the target protein\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo obtain a highly soluble form of spidroin for subsequent spinning, we constructed a \"fusion tag-intein-target protein\" system using the fusion tag NusA in combination with the intrinsic peptide Mini-Intein ∆I-CM for the expression of three kinds of chimeric spidroins with different numbers of repetitive units. In addition, the repetitive units in spidroin are believed to benefit the formation of beta-sheets and nanofibrils \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, we further investigated the changes in the secondary structure of the three kinds of chimeric spidroins during fibril formation and self-assembly. The availability of soluble spidroin allows further high-performance applications to be easily achieved. The expression and purification system developed in our study can be applied to produce proteins that are difficult to express and simplify the pretreatment process. High-performance biomimetic spidroin materials with practical applications are expected to be obtained. This study also provides theoretical references for preparing polymer materials with other complex mesoscopic structural features, such as collagen and amyloid protein.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Construction of the NusA-Intein-NTnRepCT expression system\u003c/h2\u003e \u003cp\u003eThe bacterial strains, plasmids, the sources of the gene sequences, and primers used in this study are listed in Additional File 1, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. The pET43.1a (+)-NusA-Intein-NT2RepCT expression vector used in this work was created by combining the target gene fragment (NT2RepCT) with the pET43.1a (+)-NusA-Intein vector via a double digestion-ligation (HindIII, XhoI, TAKARA) process (produced by Genewiz). (NTR2CT, NT: NCBI Accession number. AM259067; CT: NCBI Accession number JX513956; R2: NCBI Accession number. AJ973155) Overall sequence analysis was performed using ProtParam (ExPASy), and hydrophobicity analysis was performed via ProtScale. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.ExPASy.org/protscale/\u003c/span\u003e\u003cspan address=\"https://web.ExPASy.org/protscale/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe subsequent polyploid expression vector pET43.1a-NusA-Intein-NTnRepCT was generated via a head and tail strategy \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, using primers F2 and R2 (NheI and SpeI) with two compatible but non-reproducible restriction endonuclease sites (Additional File 1, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). NT2RepCT was used as a template to amplify the repeat module gene (R2), which was double digested (NheI and SpeI, TAKARA) and subsequently ligated to the single digested and expression vector pET43.1a-NusA-Intein-NT2RepCT. This process was repeated several times to obtain the fusion protein expression vector pET43.1a-NusA-Intein-NT2Rep-6RepCT with different numbers of repetitive units. A diagram of the construction process is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. To verify the role of the fusion tag NusA in enhancing the soluble expression of spidroins, three expression vectors pET43.1a-NT2Rep-6RepCT without the fusion tag NusA and intein served as a parallel control group, and the resulting recombinant plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) competent cells for transformation and a single colony was selected for PCR identification and sequenced at Genewiz (Suzhou, China).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Optimization of protein expression conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells carrying the expression plasmid (available from section 2.2) were induced with IPTG (0.1 mM) for 12 h at 30\u0026deg;C. After induction and shattering treatments, protein expression was verified by SDS-PAGE. Subsequently, the effects of inducer concentration, induction temperature, and induction time on recombinant protein expression were considered, and the specific conditions were optimized to determine the optimal conditions for protein expression. Different temperatures, such as 16\u0026deg;C, 20\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C, and 35\u0026deg;C, and different concentrations of the induction agent, 0.1 mM, 0.3 mM, 0.5 mM, and 1.0 mM, were used to induce the expression of the recombinant construct and empty vector in BL21(DE3) transformants for 6 h, 12 h, 24 h, 36 h, and 48 h, respectively. Finally, the optimal expression conditions were determined by soluble proportion and yield via SDS-PAGE. Total protein and soluble fractions were calculated by grey-scale scanning using Quantity One version 4.62 software (Bio-Rad, USA). Protein concentrations were determined using a BCA kit from Vazyme (Nanjing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Protein purification\u003c/h2\u003e \u003cp\u003eThe His-tagged recombinant proteins NT2Rep-6RepCT were purified using Ni\u003csup\u003e2+\u003c/sup\u003e-NTA agarose (QIAGEN)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The Ni2+-NTA agarose column was equilibrated with a binding buffer containing 20 mM Tris-HC l and 140 mM NaCl (pH 8.0) and eluted with an elution buffer containing 20 mM Tris-HCl, 300 mM NaCl, and 0.5 M imidazole (pH 8.0). A centrifugal ultrafiltration device (Vivaspin 20, GE Healthcare) with a molecular weight of 10 kDa was used to concentrate the spidroin and validate the analysis by SDS‒PAGE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Protein morphology observation\u003c/h2\u003e \u003cp\u003eThe purified and freeze-dried spidroin was broken by freezing in liquid nitrogen and then sputter-coated with gold on the surfaces of the proteins. The surface and cross-section of the prepared spidroins were observed under a scanning electron microscope (FESEM, Hitachi S4800, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Secondary structure analysis\u003c/h2\u003e \u003cp\u003eCircular Dichroism (CD): Recombinant spidroin conformations were determined by circular dichroism (JASCO J-1500 CD Spectrometer) in the far ultraviolet region in the wavelength range 185\u0026ndash;260 nm, and a known concentration of chimeric protein stock solution was obtained and diluted to a concentration of 0.5 mg/L. Fourier Transformed Infrared Spectroscopy (FTIR) (Bruker OPTIK GmbH Tensor II, Germany) was used to analyze the secondary structure and content of the components in the chimeric spider silk fibrils, which were dried and used for spectroscopic determination using an attenuated total reflection detector with a scanning range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a resolution of 8 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 128 scans. The spectra were processed by the software Peakfit V 4.2, and the Raman spectra in the amide I band (1700\u0026thinsp;\u0026minus;\u0026thinsp;1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were fitted with second-order derivatives to obtain the content of each component by calculating the peak area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Self-assembly ability analysis\u003c/h2\u003e \u003cp\u003eThe morphology of the chimeric arachnoid nanofibers was observed using atomic force microscopy (Bruker ICON). Before testing, the chimeric spidroin stock solution was brought to a concentration of 0.001% (w/v), and the diluted solution was subsequently dried dropwise on the surface of the mica flakes. The morphology was measured using the scanning mode and a 2 nm radius probe with a mechanical modulus of 0.4 N/m. The final nanofiber morphology and mechanical data were analyzed using NanoScope Analysis 1.8, a software program that comes with the instrument.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1. Expression of NTnRepCT spidroin (n=2, 4, and 6) and their purification\u003c/p\u003e\n\u003cp\u003eThe spidroin gene sequence contains a highly conserved N-terminal nonrepeat structural domain (N-terminal, NT), a C-terminal nonrepeat region (CT), and a core repetitive unit (REP). This study evaluated the spinning performance of the recombinant spidroins (NT2Rep-6RepCT) with different amonts of repetitive units. The recombinant spidroins constructed in this study were selected from the Rep of \u003cem\u003eEuprosthenops australis\u003c/em\u003e MaSp1, which is characterized by high amino acid levels, with Ala (A) and Gly (G) accounting for 67.8% of the total amino acids. However, this gives the nucleotide sequence a high GC content of 86%. These amino acids are primarily GGX, GX (X for other amino acids), and poly-Ala, typical of the spidroin module. The high repetitiveness of spidroin sequence poses a great challenge for its heterologous production, which results in the formation of truncated proteins, low expression, and inclusion bodies. Therefore, we constructed a \u0026quot;fusion tag-intein-spidroin\u0026quot; expression system to achieve soluble expression of spidroin. The hydrophilicity/hydrophobicity changed periodically with increasing core repetitive units and was uniformly distributed. (Additional File 1: \u003cstrong\u003eFigure. S1\u003c/strong\u003e) This amphiphilicity may also correlate with the disorganization of the spidroins, and an increase in disorganization may improve the ability of the spidroins to self-assemble. The self-assembly properties of soluble spidroins are related to the composition of their hydrophilic and hydrophobic structural domains\u003csup\u003e28\u003c/sup\u003e. Among these domains, hydrophobic structural domains are the key element in the phase separation of spidroins, which is the early stage of protein self-assembly\u003csup\u003e29\u003c/sup\u003e. The hydrophobic structural domains of spidroins are largely disordered, and the self-association of hydrophobic sequences provides the driving force for phase separation\u003csup\u003e30\u003c/sup\u003e. The greater the disorder of the protein sequence is, the stronger the intrinsic structural stability of its phase separation state, and subsequently the stronger the self-assembly ability of its protein. To obtain soluble expression of spidroin in \u003cem\u003eE. coli\u003c/em\u003e, a recombinant expression system of the fusion tag-intein-spidroin was constructed by introducing the fusion tag NusA and the intrinsic peptide Intein with self-cleaving function. A parallel control group without the fusion tag was constructed to verify the effect of the fusion tag NusA in increasing the solubility of spidroins. For subsequent purification, a histidine tag (6\u0026times;His tag) was added to the gene end (shown in\u0026nbsp;\u003cstrong\u003eFigure 1\u003c/strong\u003e). The final fusion protein containing different numbers of repetitive units in the core region of the\u0026nbsp;\u003cbr\u003espidroin \u0026quot;NusA-Intein-NTnRepCT\u0026quot; was expressed in \u003cem\u003eE. coli\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe expression of the three chimeric spidroins is shown in \u003cstrong\u003eFigure 2\u003c/strong\u003e. All three spidroins in the control group were expressed as inclusion bodies in very few amounts, and barely any bands appeared in the soluble fraction \u003cstrong\u003e(Figure 2a)\u003c/strong\u003e. Their theoretical molecular weights are approximately 32, 40, and 46 kDa, respectively. The apparent molecular weights of the fusion proteins NusA-Intein-NT2RepCT and NusA-Intein-NT4RepCT in the soluble fractions by SDS-PAGE are generally consistent with the theoretical molecular weights. In contrast, the apparent molecular weights are higher for several repetitive units of 6 (6Rep). This may be because spidroin is a special kind of self-assembling protein, which results in a high apparent molecular weight (56 kDa) due to its structure affecting its ability to migrate in SDS-PAGE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, by employing the fusion tag NusA, a small amount of expression of chimeric spidroins appeared in the soluble fraction after the self-cleavage by intein. To achieve higher expression levels, the expression conditions for the three spidroins NT2Rep-6RepCT were optimized at 0.3 mM IPTG and induced for 24 h at 20\u0026deg;C \u003cstrong\u003e(Figure 2b)\u003c/strong\u003e. On the one hand, these findings suggested that the fusion tag NusA contributes to the soluble expression of spidroin. On the other hand, for self-cleavage, a microacidic environment will be created in the \u003cem\u003eE. coli\u003c/em\u003e expression system as the secondary products gradually accumulate. In this environment, the target protein (NT2Rep-6RepCT) is produced after self-cleavage by intein to remove NusA-Intein\u003csup\u003e31\u003c/sup\u003e.\u0026nbsp;Specifically, due to the large molecular weight of NT6RepCT, self-cleavage of the NusA tag and intein may not be sufficient resulting in a large NusA-intein portion in the soluble fraction.\u0026nbsp;(\u003cstrong\u003eFigure 2b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eSubsequently, three kinds of chimeric spidroins were purified through Ni-chelating affinity chromatography. All three chimeric proteins reached a purity of over 95 % by grey-scale scanning. 266 mg/L (NT2RepCT), 135 mg/L (NT4RepCT), and 125 mg/L (NT6RepCT) were determined by the BCA method for the three purified chimeric proteins (\u003cstrong\u003eFigure 3)\u003c/strong\u003e. The yields were significantly higher compared to those reported in other literature (40 mg/L\u003csup\u003e32\u003c/sup\u003e, 125 mg/L\u003csup\u003e18\u003c/sup\u003e), thus indicating that the fusion tag NusA effectively increased the soluble expression of the three chimeric spidroins in \u003cem\u003eE. coli\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in \u003cstrong\u003eFigure 4\u003c/strong\u003e, a clear strong positive peak at around 190-195 nm and\u0026nbsp;negative peaks at around 208 and 222 nm indicated that the secondary structure of three kinds of chimeric spidroins are mainly \u0026alpha;-helix and random coil structures. This result was in good agreement with that of completely dissolved proteins (for example, recombinant spidroin\u003csup\u003e33\u003c/sup\u003e and regenerated silk protein\u003csup\u003e34\u003c/sup\u003e), suggesting a good dissolvability of these three chimeric proteins in aqueous solutions. In particular, the helical structure in these three chimeric proteins was critical to their self-assembling into ordered formations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter that, the self-assembly of chimeric spidroin was investigated by AFM observation. As shown in \u003cstrong\u003eFigure 5\u003c/strong\u003e\u003cstrong\u003e.a.c.e\u003c/strong\u003e, the three kinds of spidroin\u003cem\u003es\u003c/em\u003e were initially folded into nanoparticles with an average 4-6 nm diameter. The self-assembly of silk proteins generally followed a nucleation process, where nanoparticles provided heterogeneous sites to introduce fibrillar formation in solutions. However, the NT2RepCT could barely form nanofibrils after incubating at 60 \u0026deg;C for 48 h (\u003cstrong\u003eFigure 5b\u003c/strong\u003e), probably due to the protein\u0026apos;s lower molecular weight (only 2 of the repetitive units in the core region). The initial nanoparticles of NT2RepCT were slightly aggregated into large ones with a diameter of 10-25 nm at this condition. For comparison, the NT4RepCT and NT6RepCT with higher molecular weights presented fascinating network-like nanofibril structures at the same incubation condition (60 \u0026deg;C for 48 h), shown in \u003cstrong\u003eFigure 5\u003c/strong\u003e\u003cstrong\u003ed and 5f\u003c/strong\u003e. The diameters of these nanofibrils were approximately 6 nm, which was consistent with the diameters of their preliminary nanoparticles (\u003cstrong\u003eFigure.5c and 5e\u003c/strong\u003e). In addition, the FTIR spectra confirmed the presence of ordered structure, i.e., \u0026beta;-sheet conformation in the self-assembled proteins. As shown in Additional File 1, \u003cstrong\u003eFigure. S2\u003c/strong\u003e, the absorption peak at 1620 cm\u003csup\u003e-1\u003c/sup\u003e is assigned to the \u0026beta;-turn conformation, while the peaks at 1646 and 1675 cm\u003csup\u003e-1\u003c/sup\u003e are assigned to the random coil/helix and \u0026beta;-turn conformation, respectively. As shown in Additional File 1, \u003cstrong\u003eFigure. S3,\u003c/strong\u003e the deconvolution results of the FTIR spectra (amide I band) suggested an increase in \u0026beta;-sheet content, followed by an increasing number of repetitive units in the core region of proteins. The highest \u0026beta;-sheet content of 41.3% was presented in the NT6RepCT nanofibrils, compared to 39.1 and 38.3% in NT2RepCT and NT4RepCT samples, respectively (Additional File 1, \u003cstrong\u003eTable S2\u003c/strong\u003e). This is expected because the highly repetitive domains in protein sequences are believed to contribute to the transformation of \u0026beta;-sheets in recombinant spidroins. Notably, the self-assembly of NT4RepCT and NT6RepCT was very similar to that of native silk proteins as was the case for recombinant spidroins in other works\u003csup\u003e35,36\u003c/sup\u003e, as indicated by both the secondary structures and morphology of these nanofibrils. This self-assembly of these two proteins is promising for generating macroscale materials with robust mechanical properties.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.2. Structural characterization of NTnRepCT\u003c/p\u003e\n\u003cp\u003eThe three chimeric spidroins exhibited different aggregation morphologies after freeze-drying under the same conditions. \u003cstrong\u003eFigure 6\u003c/strong\u003e shows scanning electron microscopy (SEM) images of the resulting three spidroins, in which sample 2Rep could not form a large aggregation state due to the lack of repetitive units. This might result from the relatively small size of the repetitive core region of 2Rep spidroins (~32 kDa), which was unfavorable for initializing any fibrillar formation. However, 4Rep and 6Rep formed relatively dense interlaced and reticular structures, the surface of 4Rep spidroins had severe holes, and the aggregation of 6Rep spidroins was denser and more easily distributed. \u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion and Conclusion","content":"\u003cp\u003eKey findings are as follows: The three chimeric spidroins in this study consisted of repetitive sequences with high alanine and glycine contents, which resulted in low recombinant expression and a high tendency to form inclusion bodies. while natural spiders produce soluble spidroins in their glands. The solubility of recombinant spidroins plays a significant role in the mechanical performance of artificial fibres after subsequent spinning\u003csup\u003e17\u003c/sup\u003e. Previously,\u0026nbsp;Bowen\u0026nbsp;\u003csup\u003e12\u003c/sup\u003eand Xia et al.\u0026nbsp;\u003csup\u003e11\u003c/sup\u003eselected repeated key spidroin sequences from the natural spidroin MaSp for heterologous expression and obtained spidroins of different molecular weights expressed as inclusion bodies, which indicated the significant challenge of producing soluble recombinant spidroins that contain highly repetitive sequences, and the need to find an effective strategy for achieving soluble expression of spidroins. Here we compare the proposed strategy with conventional methods\u003csup\u003e14,37\u003c/sup\u003e, which are different from those reported previously. The fusion tag NusA, together with the self-cleavage functional Intein, effectively reduces the formation of inclusion bodies and results in the soluble expression of spidroins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy employing a self-cleavage fusion system,\u0026nbsp;the highest yield of NT2RepCT reached 266 mg/L in the study. Moreover, soluble helices facilitate subsequent spinning, and artificial silk spun from soluble spidroin may have superior mechanical properties. This established system enhanced the heterologous expression of spidroins, providing a reference for the soluble expression of difficult-to-express foreign proteins.\u003c/p\u003e\n\u003cp\u003eThe secondary structure compositions of the three kinds of chimeric spidroins with different morphologies were observed by CD and FTIR, and the trend of secondary structure transformation during morphological changes was observed. In an aqueous solution, NT2Rep-6RepCT existed mainly in the \u0026alpha;-helix conformation, but in the solid state, the \u0026beta;-sheet content was 38.3%-41.3%. By comparing the secondary structure compositions of the chimeric spider silks in aqueous solution and in the solid state, we found that the three chimeric spider silks transformed from an \u0026alpha;-helical to a \u0026beta;-sheet conformation during the drying process. A better morphology of chimeric spidroins with more repetitive units was observed by atomic force microscopy, which is consistent with the analysis of disorganization\u003csup\u003e38\u003c/sup\u003e. We consequently examined the self-assembly trends and capabilities of these materials after heat induction. The NT4RepCT and NT6RepCT,\u0026nbsp;which have higher molecular weights, presented better network-like nanofibril structures, which indicated greater self-assembly capacity. These findings support the use of localized \u0026beta;-sheet structural domains in amorphous networks by Chan et al. to increase the mechanical toughness and stability. The resulting continuous \u0026beta;-sheet nanocrystal network obtained through grafting-from polymerization exhibited greater compressive strength and stiffness than the initial network lacking \u0026beta;-sheets\u003csup\u003e39\u003c/sup\u003e. In this study, we successfully expressed the three spidroins in a soluble form and confirmed their ability to self-assemble and form fibers. However, additional verification of their spinning performance is necessary. We acknowledge that there may be significant debate among researchers regarding protein yield and spinning performance. Although Xia et al. metabolically engineered the expression host in order to modify the production of spidroins and achieved a yield of 1.2 g/L by high-density fermentation, the expression of high-molecular-weight polyploid spidroins was still accompanied by putative truncated forms of the target protein, which prevented the complete preparation of high-molecular-weight spidroins\u003csup\u003e11\u003c/sup\u003e. Later, Qian et al. used \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e as the host to establish a secretion production platform, and achieved a yield of 2.2 g/L by high-density fermentation. However, this method is limited to producing lower-molecular-weight spidroins (~40 kDa)\u003csup\u003e40\u003c/sup\u003e. Efficient secretion of high-molecular-weight spidroins via this method is challenging. Because our expression strategy proposed in this paper can provide another alternative method for the expression of spidroins, further high-density fermentation to obtain high yields of soluble spidroins and subsequent spinning validation are still l needed. In addition, our self-cleavage fusion system may serve as a reference for preparing high-performance spider silk materials for medical, military, and other fields. This approach might also lay a good foundation for the soluble expression of other proteins similar to spidroins, such as collagen and mussel proteins, and provide a theoretical reference for preparing other polymer materials with complex structures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have been involved with the work and approved the manuscript for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article [and its supplementary information files]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was performed in the absence of any commercial or financial relationships that can be considered as a potential conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBXZ: Writing \u0026ndash; original draft, Methodology. YFH: Investigation, Visualization. YQS: Conceptualization. BRA: Validation. MS: Data curation. KZ: Software, Resources. CC: Project Administration, Review \u0026amp; Editing. BFH: Funding acquisition, Supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by scientific research plan projects of Shaanxi education department (22JC010), and Key R\u0026amp;D general projects of Shaanxi provincial department of science and technology (2023-YBNY-170), Natural science foundation of China (81973531), and the fundamental research project of the Shenzhen science and technology innovation commission (20200812211704001).\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLewis, R. V. Spider Silk: Ancient Ideas for New Biomaterials. \u003cem\u003eChem Rev\u003c/em\u003e \u003cstrong\u003e2006\u003c/strong\u003e, \u003cem\u003e106\u003c/em\u003e (9), 3762\u0026ndash;3774. https://doi.org/10.1021/cr010194g.\u003c/li\u003e\n\u003cli\u003eTokareva, O.; Jacobsen, M.; Buehler, M.; Wong, J.; Kaplan, D. L. Structure-Function-Property-Design Interplay in Biopolymers: Spider Silk. \u003cem\u003eActa Biomater\u003c/em\u003e \u003cstrong\u003e2014\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e (4), 1612\u0026ndash;1626. https://doi.org/10.1016/j.actbio.2013.08.020.\u003c/li\u003e\n\u003cli\u003eBlackledge, T. A.; Summers, A. P.; Hayashi, C. Y. 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Secretory Production of Spider Silk Proteins in Metabolically Engineered Corynebacterium Glutamicum for Spinning into Tough Fibers. \u003cem\u003eMetab Eng\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e70\u003c/em\u003e, 102\u0026ndash;114. https://doi.org/10.1016/j.ymben.2022.01.009.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"chimeric spider silk protein, fusion tag, self-cleavage intein, soluble expression","lastPublishedDoi":"10.21203/rs.3.rs-3905454/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3905454/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe spider silk protein (spidroin), which has powerful mechanical properties, has been extensively studied and shown potential application in various fields. The predatory nature of spiders makes native spidroin challenging to obtain, while heterologous expression of spidroin was hindered by the gene sequence features such as highly repetitive regions and high GC content. The low yield of spidroin subsequently affects its further application. In this study, we constructed a convenient expression system by employing a fusion tag in combination with a self-cleavage intronic peptide (intein) for three kinds of chimeric spidroins with different numbers of repetitive units, and soluble expression of the three kinds of spidroins after optimizing expression conditions was achieved with yields of 266 mg/L (NT2RepCT), 135 mg/L (NT4RepCT), and 125 mg/L (NT6RepCT), respectively. Three kinds of chimeric spidroins displayed increased β-sheet content with increased repetitive units during the transition from the solution to the dry state. Their capacity to form filamentous fibrils increased with the number of repetitive numbers. This study provides a solution for spidroin soluble expression and lays a foundation for its future application.\u003c/p\u003e","manuscriptTitle":"Engineering self-cleavage fusion system for the Production of Chimera Spider Silk Proteins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-06 16:30:17","doi":"10.21203/rs.3.rs-3905454/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e165823b-86f8-4204-b7f8-399308c82acd","owner":[],"postedDate":"February 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-15T10:16:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-06 16:30:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3905454","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3905454","identity":"rs-3905454","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}