Recombinant prokaryotic expression vector, expression strain, soluble human protein and application

Abstract

The invention discloses a recombinant prokaryotic expression vector, an expression strain, soluble human-derived protein and application, the recombinant prokaryotic expression vector comprises a first nucleotide sequence for coding a target human-derived protein and a second nucleotide sequence for coding a cold-adapted molecular chaperone, and a rare codon corresponding to host bacteria is introduced into the first nucleotide sequence to obtain a recombinant prokaryotic expression vector. The translation rate of a key site is actively reduced, a cold-adapted molecular chaperone is introduced into a second nucleic acid sequence, and correct folding and soluble expression of the human-derived protein in a prokaryotic host are promoted in a low-temperature induction manner, so that the high-yield and high-solubility human-derived soluble protein is obtained. The invention provides a feasible way for large-scale preparation of human soluble protein, and has important industrial application value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein production technology, specifically to a recombinant prokaryotic expression vector, expression strain, soluble human protein and its uses, and particularly to a system and method for efficiently producing soluble human protein in prokaryotic hosts such as Escherichia coli through the synergistic effect of low-temperature induction, translation rate regulation and cold-adapted molecular chaperone co-expression. Background Technology

[0002] Prokaryotic expression systems (especially *E. coli*) offer significant advantages in producing recombinant human proteins, including low cost, short cycle time, and high yield. However, many human proteins, when expressed in prokaryotic hosts, readily form insoluble inclusion bodies due to the lack of eukaryotic-specific post-translational modification systems, differences in molecular chaperone environments, and varying codon usage preferences. This leads to protein inactivation, complex subsequent refolding processes, and low yields. Collagen, a major structural protein in animal connective tissue, has wide applications in biomedicine and tissue engineering. Type III collagen is primarily found in skin and blood vessels, and is closely related to skin elasticity and wound healing. Compared to collagen extracted from animal tissues, recombinant human collagen offers advantages such as low immunogenicity and no pathogen risk. However, efficient expression of human collagen in prokaryotic systems like *E. coli* presents significant challenges. Overexpression of heterologous proteins in *E. coli* often results in misfolding and the formation of insoluble inclusion bodies. Collagen, with its large molecular weight and rich glycine-proline repeat sequences, readily aggregates in the cytoplasm. Furthermore, codon bias differences in human genes within *E. coli* can also affect expression efficiency and correct folding. For example, translation pauses at certain codons are necessary for correct folding; replacing these codons with high-frequency synonymous codons can lead to overly rapid translation, potentially increasing protein misfolding. Therefore, efficient direct expression of human type III collagen genes in *E. coli* often yields large amounts of insoluble products with very low yields of functional proteins.

[0003] To address this issue, existing technologies typically employ a single optimization strategy, such as: (1) using low-temperature culture to slow down protein synthesis and promote correct folding, but simply cooling has limited effect on promoting the folding of complex proteins; (2) optimizing the codons of the target protein gene by replacing rare codons with host-preferred codons to improve translation efficiency, but this may exacerbate aggregation due to excessively fast synthesis; (3) co-expressing molecular chaperones (such as GroEL / ES, DnaK / DnaJ systems) to assist folding, but classic molecular chaperone systems are usually highly induced under heat shock conditions, and their expression levels and activities may be insufficient at low temperatures. Currently, there are no reports of molecular chaperones specifically designed for low-temperature environments for the folding of human recombinant soluble proteins, especially collagen.

[0004] Therefore, there is an urgent need in this field for a comprehensive solution that can systematically address the problems of low solubility, inability to fold correctly, and easy formation of inclusion bodies in prokaryotic expression of human proteins, in order to obtain high-yield and soluble recombinant human proteins, especially human type III collagen, in prokaryotic cells. Summary of the Invention

[0005] This invention aims to overcome the aforementioned deficiencies of existing technologies and provide a comprehensive solution that can significantly improve the soluble expression yield of human proteins in prokaryotic systems. The core concept of this invention lies in breaking away from conventional single-optimization approaches and creatively integrating low-temperature induction, active deceleration of the translation process, and co-expression of cold-adaptive molecular chaperones to create a microenvironment within prokaryotic cells optimal for the correct folding of human proteins. The technical solution is as follows: In a first aspect, the present invention provides a recombinant prokaryotic expression vector for producing soluble human proteins, the expression vector comprising a recombinant expression construct encoding a first nucleic acid sequence encoding the human protein and a second nucleic acid sequence encoding a cold-adaptive molecular chaperone, wherein... The first nucleic acid sequence includes at least one rare codon mutation site corresponding to the host bacterium, in order to reduce the translation rate of the human protein in the prokaryotic expression system; The second nucleic acid sequence may encode at least one cold-adaptive molecular chaperone and induce the expression of the first nucleic acid sequence at temperatures below 30°C.

[0006] Preferably, the first and second nucleic acid sequences are located on the same plasmid, or on two different plasmids, or at least one of them is integrated into the chromosome.

[0007] Preferably, the human-derived protein includes at least one of IFN-α / β / γ, IL-2, IL-6, IL-11, G-CSF, EGF, bFGF, type III human collagen, insulin, glucagon-like peptide-1, brain natriuretic peptide, superoxide dismutase, trypsin, deintegrin metalloproteinase catalytic domain, tumor necrosis factor receptor, human growth hormone, leptin, serum albumin, Fab, scFv, VHH, G protein-coupled receptor extracellular domain, and CD4 extracellular domain.

[0008] Preferably, the human-derived protein is type III human collagen, corresponding to the cDNA sequence encoding the type III human collagen, and the rare codon mutation site is located at the boundary of the encoding Gly-Pro-Hyp segment and / or in a region where local structure formation is likely to occur.

[0009] Preferably, corresponding to the cDNA sequence encoding the type III human collagen, the rare codon mutation site mutates at least one CGT codon from 703-705, 1237-1239, 1714-1716, 2191-2193, 2866-2868, 3325-3327, 3778-3780, and 4294-4296 to AGA or AGG.

[0010] Preferably, the cold-adaptation molecular chaperone includes cold shock proteins, cold-adaptation chaperone protein complexes, cold-adaptation folding enzymes, or functional combinations thereof. Preferably, the cold-adaptation molecular chaperone expresses GroEL / ES or DnaK and DnaJ proteins from psychrophilic polar sea ice isolates.

[0011] In a second aspect, the present invention provides a recombinant expression strain comprising the recombinant prokaryotic expression vector for producing soluble human proteins as described in any of the above embodiments.

[0012] Preferably, the host bacteria of the original expression strain include at least one of Escherichia coli, Bacillus subtilis, and lactic acid bacteria.

[0013] Compared with the prior art, the beneficial effects that at least one technical solution adopted in the embodiments of this specification can achieve include at least: First, this invention introduces at least one rare codon mutation site corresponding to the host bacterium into the first nucleic acid sequence encoding a human soluble protein. The amino acid sequence encoded by this mutation site corresponds to a key site in the three-dimensional structure of the human soluble protein. This reduces the translation rate of the human protein in the prokaryotic expression system. For example, for the cDNA sequence of type III human collagen, this invention creatively replaces eight key arginine (Arg) CGT codons with codons rarely used in E. coli (such as AGG and AGA). The introduction of these rare codons can cause pauses in translation within prokaryotic cells, thereby giving the peptide chain more time to fold and reducing erroneous conformations. This "codon deoptimization" strategy effectively reduces the transient synthesis rate and promotes the formation of the correct triple helix structure.

[0014] Second, the present invention can still induce prokaryotic cells to express human soluble proteins at low temperatures below 30°C. Lowering the culture temperature can significantly reduce misfolding and inclusion body formation of recombinant proteins, allowing more target proteins to fold in their native conformation.

[0015] Third, this invention introduces cold-adaptive molecular chaperone co-expression, which can still efficiently perform chaperone functions at low temperatures (below 30°C), providing an auxiliary environment for the folding of human proteins and preventing mis-aggregation between chains. Compared to the chaperone naturally expressed by *E. coli*, this system is more suitable for low-temperature culture conditions. For the type III human collagen expressed in the embodiments of this invention, the molecular chaperone DnaK / DnaJ derived from the polar sea ice strain *Pseudoalteromonas* is co-expressed simultaneously with collagen expression. This psychrophilic bacterium, DnaK / DnaJ, possesses structural cold-adaptive characteristics, enabling it to efficiently perform chaperone functions even at low temperatures.

[0016] Fourth, through the above three-pronged approach, this invention enables the production of high-yield, highly soluble human-derived proteins in prokaryotic cells such as *Escherichia coli*. Verification with recombinant type III human collagen showed that almost all target proteins existed in soluble form with virtually no inclusion body precipitation. This method provides a feasible route for the large-scale preparation of human-derived soluble proteins and has significant industrial application value. The recombinant type III human collagen *Escherichia coli* PE2501-hCo1 strain was deposited on January 28, 2026, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026275, located at Wuhan University, Wuhan, China.

[0017] Fifth, in the co-expression system of the present invention, the first nucleic acid sequence expressing the human protein and the second nucleic acid sequence expressing the cold-adapted molecular chaperone can be located on the same plasmid or on two different plasmids, or at least one of them can be integrated into the chromosome, and more preferably inserted into two different expression plasmid vectors, thereby making the expression level easier to control and the induction control more flexible. Attached Figure Description

[0018] Figure 1 The image shown is an SDS-PAGE gel image of recombinant human collagen of the present invention. Lane 1: Whole cell lysate band without IPTG and L-arabinose induction; Lane 2: Whole cell lysate band after induction of expression; Lane 3: Precipitate (insoluble matter) band after cell centrifugation; Lane 4: Supernatant (soluble protein) band after cell centrifugation; Lane 5: Breakthrough peak band that was not adsorbed by the packing column and flowed directly out with the mobile phase after induction of expression; Lane 6: Wash flow-through band after washing buffer passes through the column; Lane 7: Protein band obtained after elution and purification. Figure 2 The results of circular dichroism (CD) detection of recombinant human collagen of the present invention are shown. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, the scope of protection of the present invention is not limited thereto. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Unless otherwise specified, the drugs and reagents involved in the embodiments are all ordinary commercially available products; unless otherwise specified, the experimental operations involved in the embodiments are all conventional operations in the art. Unless otherwise specified, the percentages involved in the embodiments are all mass percentages.

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] It should be noted that the terms "first," "second," etc., in the specification, claims, and drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion.

[0022] This invention provides a recombinant prokaryotic expression vector for producing soluble human proteins. The expression vector includes a first nucleic acid sequence encoding the human protein. Preferably, the human protein is selected from at least one of the following: IFN-α / β / γ, IL-2, IL-6, IL-11, G-CSF, EGF, bFGF, type III human collagen, insulin, glucagon-like peptide-1, brain natriuretic peptide, superoxide dismutase, trypsin, detegrin metalloproteinase catalytic domain, tumor necrosis factor receptor, human growth hormone, leptin, serum albumin, Fab, scFv, VHH, G protein-coupled receptor extracellular domain, and CD4 extracellular domain. In the following description, we will use type III human collagen as an example, but it should be understood that type III human collagen is only a preferred example of the human protein of this invention and should not be construed as limiting the scope of the claims. To reduce the translation rate of the human protein in prokaryotic cells, especially at key sites in the protein's three-dimensional structure, such as α / β... The translation rate of amino acids in the following regions is affected: structural domains, disulfide-rich structural domains, key regions of long-range interactions, sequences containing consecutive or adjacent cysteine ​​residues, proline residues near turn or loop regions, sequences preceding the coding sequence of cofactor binding pockets or metal ion coordination sites, or the boundary region of glyco-pro-hydroxypro (tripeptide repeat) segments. In this embodiment, rare codon mutation sites corresponding to the host bacteria are set at the first nucleic acid sequence position corresponding to at least one of the above-mentioned key sites in the three-dimensional structure of the protein to reduce the translation rate of the human protein at the above-mentioned rare mutated codon sites. The introduction of these rare codons can cause pauses in the translation process in prokaryotic cells, thereby giving the peptide chain more time to fold and reducing incorrect conformations. This "codon deoptimization" strategy effectively reduces the transient synthesis rate and promotes the formation of the correct triple helix structure.

[0023] The expression vector further includes a second nucleic acid sequence encoding a cold-adaptive molecular chaperone, which can induce the correct expression and folding of the first nucleic acid sequence at temperatures below 30°C. The cold-adaptive molecular chaperone includes, but is not limited to, cold shock proteins, cold-adaptive chaperone protein complexes, cold-adaptive folding enzymes, or functional combinations thereof, specifically selected from GroEL / ES, DnaK / DnaJ proteins from psychrophilic polar sea ice isolates, psychrophilic bacterial Cpn60 (such as PhCpn60), and psychrophilic Shewanella (…). Shewanella oneidensis Cold-adapted J-domain co-chaperone protein system, Antarctic ice yeast ( Glaciozyma antarcticaAt least one of the TRiC / CCT chaperone proteins from psychrophilic yeast. These cold-adaptive molecular chaperone proteins possess cold-adaptive characteristics, maintaining high conformational flexibility and catalytic efficiency at low temperatures. This invention introduces cold-adaptive molecular chaperones for co-expression, enabling them to efficiently perform chaperone functions at low temperatures (below 30°C), providing an auxiliary environment for the folding of human proteins and preventing mis-aggregation between chains.

[0024] In this invention, the first and second nucleic acid sequences can be inserted into the same expression plasmid vector. The two genes replicate and are inherited synchronously as a unit, making them less prone to loss or ratio changes, resulting in high genetic stability. However, gene expression flexibility is low and difficult to regulate, with the copy number ratio of the two genes fixed at 1:1. Furthermore, this invention can also integrate at least one of the first and second nucleic acid sequences into a prokaryotic cell genome to achieve stable inheritance, allowing for the construction of engineered strains for long-term use. However, this also faces the problem of low gene expression flexibility and difficulty in regulation. Therefore, most preferably, the first and second nucleic acid sequences are inserted into two different expression plasmid vectors. Although the two plasmids may be unevenly distributed during replication and division, easily leading to the loss of one and resulting in low genetic stability, their gene expression regulation flexibility is high. The expression levels of each can be independently regulated in two dimensions by selecting plasmids with different copy numbers or using promoters of different strengths. This is more suitable for scenarios where this invention requires independent optimization or regulation of the expression levels of two proteins for expression condition screening. Those skilled in the art can choose one of the above three methods as a suitable gene co-expression method according to actual needs.

[0025] This invention also provides a recombinant expression strain, comprising the recombinant prokaryotic expression vector for producing soluble human proteins as described in any of the above embodiments, wherein the host bacteria of the recombinant expression strain include at least one of *Escherichia coli*, *Bacillus subtilis*, and *Lactobacillus*. Using this recombinant expression strain, this invention can also induce the expression of the cold-adaptive molecular chaperone at temperatures below 30°C, and induce the correct expression and folding of human soluble proteins under the synergistic effect of the cold-adaptive molecular chaperone. Furthermore, prokaryotic cells can still be induced to express human soluble proteins at temperatures below 30°C. Lowering the culture temperature can significantly reduce misfolding and inclusion body formation of recombinant proteins, allowing more target proteins to fold in their native conformation. This invention achieves high yields and high soluble human soluble proteins in prokaryotic cells such as Escherichia coli through the synergistic effects of "low-temperature induction," "translation rate regulation," and "cold-adaptive molecular chaperone co-expression," with almost no inclusion body precipitation. This provides a feasible route for large-scale preparation of human soluble proteins and has significant industrial application value. At the same time, since most of the target proteins expressed are folded in their native conformation, it lays the foundation for their application in fields such as biomedicine, tissue engineering, and cosmetic surgery.

[0026] The following detailed description uses type III human collagen as a specific example. The recombinant type III human collagen Escherichia coli PE2501-hCo1 strain was deposited on January 28, 2026, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026275, located at Wuhan University, Wuhan, China. However, those skilled in the art should understand that this example is merely a preferred illustration of the human protein of this invention and should not be construed as limiting the scope of the claims. Furthermore, the experimental conditions and parameters of this example are closely related to the selection of plasmid expression vectors, cold-adapted molecular chaperones, and human proteins. The experimental conditions and plasmid vector types involved in this example cannot fully cover the expression of other human proteins and cold-adapted molecular chaperones.

[0027] Example 1. Construction of expression vector Target gene: The coding sequence for human type III collagen (e.g., cDNA derived from the human COL3A1 gene, NCBI ReferenceNM_000090.4) was selected, appropriately truncated to remove the signal peptide and propeptide, and codon optimized to suit prokaryotic expression. To improve folding efficiency, this embodiment performed special processing on the sequence: eight arginine codons were replaced with rare codons from *E. coli*. These eight sites were selected in key regions of the collagen amino acid sequence, such as the boundaries of the glyphosate-proline (tripeptide repeat) segment or regions prone to local structural formation. The specific sites and variations of these rare codon mutations are shown in the table below. These rare codon mutations can be achieved using at least one of the conventional primer PCR method, in vivo homologous recombination method, or enzyme digestion-ligation method. The final genetically engineered human type III collagen cDNA sequence is shown in SEQ ID NO.1, where the rare codon mutation sites are described in Table 1 below. Introducing these rare codon mutation sites can introduce short pauses during translation, promoting correct local folding.

[0028] Table 1 Expression vector: pET28a(+) was used as the expression vector. This vector contains a T7 promoter and an IPTG induction mechanism, and the resistance marker is kanamycin. To avoid interference from additional tags during folding, the His tag sequence in the multiple cloning site of the expression vector was removed during construction. The target gene was inserted using NdeI / XhoI restriction sites to ensure that the reading frame does not contain a His-tag coding sequence. The recombinant plasmid pET28a-COL3 was thus constructed. This plasmid can efficiently express the target collagen in *E. coli* BL21(DE3) after IPTG induction. The target collagen expression product does not contain a fusion tag, facilitating the direct acquisition of high-purity collagen.

[0029] Companion vector: The plasmid pCold-DKJ for DnaK / DnaJ co-expression was constructed. This plasmid uses the pACYC series as its backbone (p15A replicon, chloramphenicol resistant) and is compatible with pET28a. In this embodiment, the DnaK and DnaJ genes (their gene sequences are shown in NCBI References DQ504163.1 and DQ640312.1) from *Pseudoalteromonas* sp. (polar sea ice isolate) were cloned into the multiple cloning site of this vector, forming an operon co-expression under promoter control. Furthermore, the araBAD promoter was modified in pCold-DKJ to control dnaKJ gene expression, making it inducible with L-arabinose. This allows for the addition of arabinose when needed to simultaneously induce high-level expression of the chaperone protein. The pCold-DKJ plasmid constructed above encodes the DnaK and DnaJ proteins from the psychrophilic Pseudoalteromonas family. Its amino acid sequence contains typical Hsp70 / DnaK family conserved domains. These chaperone proteins exhibit cold-adaptive characteristics, maintaining high conformational flexibility and catalytic efficiency at low temperatures. The plasmid replicates stably in *E. coli* and does not conflict with the pET28a-COL3 plasmid, making it suitable for chaperone co-expression.

[0030] Example 2. Expression strain and culture conditions Strain selection: *Escherichia coli* BL21(DE3) was used as the expression host. This strain carries the DE3 lysate fragment, which can efficiently transcribe the target gene COL3 on the pET28a vector via T7 RNA polymerase in the presence of IPTG. Furthermore, BL21 has low background protease activity, which is conducive to the stable accumulation of the target protein.

[0031] Culture medium: Initially, LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) was used. Alternatively, at the fermentation level, a richer 2×YT medium or a self-prepared fermentation medium (such as adding glycerol, yeast extract, and inorganic salts to increase cell density) could be used. 50 µg kanamycin and 34 µg chloramphenicol were added per liter of medium to maintain the plasmid.

[0032] Induction strategy: The engineered strain was first cultured at 37°C with shaking until the mid-log phase (OD2). 600 ≈0.6–0.8). At this point, lower the culture temperature from 37°C to 25°C and add the inducing agents: IPTG (final concentration 0.5 mM, for inducing collagen expression in pET28a-COL3) and L-arabinose (final concentration 0.2%, for inducing chaperone protein expression in pCold-DKJ). Induction culture at low temperature for approximately 8–12 hours. Lowering the temperature significantly increases the chance of correct protein folding. Since the bacterial growth rate slows down after the temperature decreases, the induction time can be extended overnight (e.g., 16 hours) to obtain higher yields. It is important to note that adding IPTG / arabinose after switching the temperature to 25°C helps avoid excessively rapid initial synthesis of the target protein. Maintain adequate aeration of the culture medium throughout the induction process (250 rpm shaking or aeration and stirring in the fermenter) to prevent insufficient dissolved oxygen at low temperatures.

[0033] Molecular chaperone expression: Following the above strategy, DnaK / DnaJ expression is initiated almost synchronously with collagen production. Upon addition of arabinose, DnaK / DnaJ are synthesized in large quantities under the drive of the araBAD promoter. These chaperones are highly active at 25°C, binding nascent peptides during peptide chain synthesis to prevent mis-aggregation and guide correct folding. Therefore, this approach, through the timely assistance of chaperones, captures most of the collagen chains and helps them form the correct triple helix.

[0034] Example 3. Analysis and Validation of the Expression Product After the induction culture is completed, the bacterial cells are collected for analysis: Cell lysis and soluble / insoluble analysis: An equal volume of bacterial pellet was taken and resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, etc., with the possible addition of protease inhibitor PMSF). Cells were sonicated in an ice bath to prevent collagen degradation. The lysis buffer was centrifuged at 12,000 × g for 15 minutes to separate the supernatant (soluble protein fraction) from the pellet (insoluble matter, including possible inclusion bodies). Equal volumes of supernatant and pellet (the latter resuspended in an equal volume of buffer) were taken for SDS-PAGE analysis. Results are shown below. Figure 1As shown, Lane 1: whole cell lysate band without IPTG and L-arabinose induction; Lane 2: whole cell lysate band after induction; Lane 3: precipitate (insoluble matter) band after cell centrifugation; Lane 4: supernatant (soluble protein) band after cell centrifugation; Lane 5: breakthrough peak band that was not adsorbed by the packing column and flowed directly out with the mobile phase after induction; Lane 6: wash flow-through band after washing buffer passes through the column; Lane 7: protein band obtained after elution and purification.

[0035] The results showed that after induction with IPTG and L-arabinose, a distinct new protein band appeared in Lane 2 (indicated by the arrow). This band was almost absent in the precipitate sample of Lane 3, but strongly visible in the supernatant sample of Lane 4, indicating that most of the recombinant collagen existed in soluble form in the cell lysate. These results demonstrate that the technical solution of this invention successfully achieved the expression of a large amount of human type III soluble collagen: the target protein band appeared in the crude cell extract after induction, mainly distributed in the soluble fraction, accounting for a large proportion of the total protein. In contrast, the precipitate fraction showed almost no corresponding band, proving that inclusion body formation was significantly inhibited.

[0036] Yield estimation: By comparing with protein standards of known concentrations, the yield of recombinant collagen in the supernatant can reach several hundred milligrams per liter of culture supernatant. For example, in 500 mL shake flask culture, the yield of soluble type III collagen in the supernatant reaches approximately 0.2–0.3 g / L. Further scaling up to high-density culture in fermenters is expected to significantly increase the yield.

[0037] Purification and Identification: Since this recombinant protein does not carry a His tag, purification can be achieved using a combination of salting-out and gel filtration. Collagen's solubility decreases under high concentrations of NaCl (e.g., 2 M NaCl), causing it to precipitate. Salting-out can be used to enrich the target protein and remove impurities. The resuspended protein is then subjected to size exclusion chromatography to obtain high-purity collagen monomers. Figure 1 As shown in Lane 7, the purified product was re-examined by SDS-PAGE, showing a single band. The semi-quantitative results calculated by grayscale showed that the purified collagen had a purity greater than 90%.

[0038] Functional conformation verification: To confirm whether the recombinant collagen folded correctly to form a triple helix structure, we further used circular dichroism (CD) to perform spectral analysis on the purified protein.

[0039] The far-UV CD spectrum was measured in phosphate buffer at 20°C and pH 7.5, and the results are as follows: Figure 2As shown, the spectrum exhibits a typical triple helix CD signal of collagen: a small positive peak at approximately 220 nm, a zero light transmission point at 213–215 nm, and a deep negative peak at approximately 200 nm. This spectral shape is highly consistent with the triple helix structure of natural collagen: the position and intensity of the positive / negative peaks indicate that the protein is rich in the secondary structure of left-handed polypeptide type II helices (i.e., collagen helices). Therefore, the product of this invention matches natural collagen in both secondary and tertiary structures, suggesting that its biological functions, such as cell adhesion and support, should also be close to those of natural collagen.

[0040] In summary, this invention, through improvements in three aspects—low-temperature deceleration expression, codon adjustment, and cold adaptation chaperone assistance—successfully yielded high-yield, soluble, and correctly folded human soluble proteins in a prokaryotic cell expression system. This method solves the long-standing problem of obtaining soluble products from prokaryotic expression of human soluble proteins. The produced human proteins can be used in the development of biomedical materials, such as the preparation of tissue engineering scaffolds, wound dressings, or cosmetic formulations, and have broad application prospects.

[0041] In this specification, the same or similar parts between the various embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, the descriptions of the embodiments described later are relatively simple, and relevant parts can be referred to the descriptions of the foregoing embodiments.

[0042] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A recombinant prokaryotic expression vector for producing soluble human proteins, characterized in that, The expression vector comprises a recombinant expression construct encoding a first nucleic acid sequence encoding the human protein and a second nucleic acid sequence encoding a cold-adaptation molecular chaperone, wherein, The first nucleic acid sequence includes at least one rare codon mutation site corresponding to the host bacterium, in order to reduce the translation rate of the human protein in the prokaryotic expression system; The second nucleic acid sequence may encode at least one cold-adaptive molecular chaperone and induce the expression of the first nucleic acid sequence at temperatures below 30°C.

2. The recombinant prokaryotic expression vector for producing soluble human proteins according to claim 1, characterized in that, The first and second nucleic acid sequences are located on the same plasmid, or on two different plasmids, or at least one of them is integrated into the chromosome.

3. The recombinant prokaryotic expression vector for producing soluble human proteins according to claim 1, characterized in that, The human-derived proteins include at least one of IFN-α / β / γ, IL-2, IL-6, IL-11, G-CSF, EGF, bFGF, type III human collagen, insulin, glucagon-like peptide-1, brain natriuretic peptide, superoxide dismutase, trypsin, detegrin metalloproteinase catalytic domain, tumor necrosis factor receptor, human growth hormone, leptin, serum albumin, Fab, scFv, VHH, G protein-coupled receptor extracellular domain, and CD4 extracellular domain.

4. The recombinant prokaryotic expression vector for producing soluble human proteins according to claim 3, characterized in that, The human-derived protein is type III human collagen, corresponding to the cDNA sequence encoding the type III human collagen. The rare codon mutation sites are located at the boundary of the Gly-Pro-Hyp segment and / or in regions where local structures are prone to form.

5. The recombinant prokaryotic expression vector for producing soluble human proteins according to claim 4, characterized in that, Corresponding to the cDNA sequence encoding the type III human collagen, the rare codon mutation site mutates at least one of the CGT codons 703-705, 1237-1239, 1714-1716, 2191-2193, 2866-2868, 3325-3327, 3778-3780, and 4294-4296 to AGA or AGG.

6. The recombinant prokaryotic expression vector for producing soluble human proteins according to any one of claims 1-5, characterized in that, The cold-adaptation molecular chaperones include cold shock proteins, cold-adaptation chaperone protein complexes, cold-adaptation folding enzymes, or functional combinations thereof.

7. The recombinant prokaryotic expression vector for producing soluble human proteins according to claim 6, characterized in that, The cold-adaptation molecular chaperone expresses GroEL / ES or DnaK and DnaJ proteins from psychrophilic polar sea ice isolates.

8. A recombinant expression strain, characterized in that, Including the recombinant prokaryotic expression vector for producing soluble human proteins as described in any one of claims 1-7.

9. The recombinant expression strain according to claim 8, characterized in that, The host bacteria of the original expression strain include at least one of Escherichia coli, Bacillus subtilis, and lactic acid bacteria.

Images