Optimized gene of full-length human-derived III-type collagen, expression system, preparation method and application

Abstract

The invention discloses a recombinant full-length human-derived III-type collagen as well as a preparation method and application of the recombinant full-length human-derived III-type collagen. The method comprises the following steps: replacing at least one corresponding rare codon of glycine, proline, arginine, leucine, isoleucine, valine and / or glutamic acid host bacteria in a Gly-Pro-X or Gly-X-Pro tripeptide structure coded by a wild full-length human III-type collagen gene sequence with a synonymous codon used by the host bacteria at high frequency; the translation efficiency in prokaryotic systems such as escherichia coli is obviously improved; the method comprises the following steps: designing 6-12 gene segments with overlapping sequences, splicing by adopting an overlapping extension PCR (Polymerase Chain Reaction) technology to obtain a full-length optimized gene, constructing the full-length optimized gene to an expression vector, and transforming the full-length optimized gene into host bacteria for induced expression; the expression product exists in the form of an inclusion body, and the natural conformation of the expression product is recovered through in-vitro renaturation; according to the method, high-efficiency expression and active preparation of the full-length human-derived III-type collagen in a prokaryotic system are realized.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein expression technology, specifically to an optimized full-length human type III collagen-encoding nucleic acid molecule, its synthesis method, a recombinant expression system containing the nucleic acid molecule, the recombinant protein expressed therefrom, and its use in tissue engineering and cosmetic surgery. Background Technology

[0002] Human type III collagen is an important structural protein of connective tissue, abundant in tissues such as skin and blood vessels, and plays a crucial role in maintaining skin elasticity, promoting wound healing, and tissue repair. Its natural sources are limited, extraction is difficult, and there is a risk of pathogen contamination. Although expressing recombinant human type III collagen in hosts such as E. coli using genetic engineering techniques has become a research hotspot, its long gene sequence and the inclusion of codons uncommon or rare in prokaryotic systems such as E. coli result in low expression efficiency and low protein yield, hindering industrial-scale production. Existing research literature generally points out that the expression of full-length collagen in prokaryotic systems is extremely difficult. For example, Shoulders and Raines pointed out in their review article "Collagen Structure and Stability" (Annual Review of Biochemistry, 2009) that due to the typical Gly-XY tripeptide repeat structure and complex triple helix folding process of collagen, intact collagen is usually difficult to fold correctly in prokaryotic expression systems. Olsen et al. also pointed out in "Recombinant collagen and gelatin for biomedical applications" (Advanced Drug Delivery Reviews, 2003) that the expression of intact collagen in E. coli is extremely challenging due to the large molecular weight of collagen and the need for a complex folding mechanism. Therefore, researchers typically employ a truncated sequence expression strategy. CN111087463B discloses a recombinant human type III collagen and its prokaryotic expression method, which selects a functional domain (1230 bp) of type III collagen as the recombinant protein expression target. The reason for choosing a truncated mutant is that only short-sequence collagen is more soluble. Therefore, in existing technologies, the collagen expressed in E. coli is short-sequence soluble recombinant collagen, which can be harvested from the supernatant. The purpose of their codon optimization is to express and recover soluble recombinant collagen more efficiently. However, there is no literature documenting the expression of full-length human type III collagen because it produces a large number of inclusion bodies, which are usually considered to be the result of expression failure or misfolding. In addition, collagen has a large molecular weight and contains a large number of glycine-proline repeat sequences, which are prone to misfolding in prokaryotic systems such as E. coli to form insoluble inclusion bodies. In existing technologies, the synthesis and splicing of long gene fragments also often face the problems of cumbersome steps and low success rate.

[0003] Therefore, it is necessary to develop an optimized gene capable of efficiently expressing human type III collagen and an in vitro refolding method for its expressed protein, which has significant industrial value. Summary of the Invention

[0004] This invention aims to overcome the aforementioned deficiencies of the prior art. Its primary objective is to provide a codon-optimized nucleic acid molecule suitable for efficient expression in prokaryotic hosts (especially *E. coli*). Its second objective is to provide a recombinant expression vector containing this nucleic acid molecule and a recombinant expression strain. Its third objective is to provide a method for producing recombinant human type III collagen using the aforementioned strain, and the resulting recombinant protein with a natural or near-natural conformation. A further objective is to elucidate the specific applications of this recombinant expression strain in tissue engineering and cosmetic surgery. The technical solution is as follows: In a first aspect, the present invention provides a nucleic acid molecule encoding recombinant full-length human type III collagen, wherein the nucleic acid molecule is codon-optimized by replacing at least one rare codon of the host bacterium corresponding to the Gly-Pro-X or Gly-X-Pro tripeptide structure in the wild-type full-length human type III collagen gene sequence with a synonym codon frequently used by the host bacterium, while maintaining the amino acid length and sequence unchanged.

[0005] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.1.

[0006] In a second aspect, the present invention provides a recombinant expression vector containing the nucleic acid molecules described in the above embodiments.

[0007] In a third aspect, the present invention provides a recombinant expression strain comprising the recombinant expression vector described in the above embodiments.

[0008] Preferably, the host bacteria of the recombinant expression strain include at least one of Escherichia coli, Bacillus subtilis, and lactic acid bacteria.

[0009] In a fourth aspect, the present invention provides the use of the recombinant expression strains of the above embodiments in the fields of tissue engineering and cosmetic surgery.

[0010] The present invention provides a method for synthesizing recombinant full-length human type III collagen nucleic acid molecules in a fifth aspect, comprising the following steps: S1. Codon optimization design: Replace at least one glycine, proline, arginine, leucine, isoleucine, valine and / or glutamic acid codon corresponding to the Gly-Pro-X or Gly-X-Pro tripeptide structure in the wild-type full-length human type III collagen gene sequence with a synonym codon frequently used by the host bacteria to design an improved full-length human type III collagen gene. S2. Synthesis of improved full-length human type III collagen gene: The improved human type III collagen gene is divided into 6-12 fragments, and each adjacent fragment is designed with a 15-20 bp overlapping sequence region. The above 6-12 fragments are synthesized sequentially by artificial synthesis. S3. Primer design: Design specific primer sequences for the overlapping sequence regions of each of the fragments; S4. Fragment splicing: Each fragment is amplified separately using primers containing the overlapping sequence region. Then, adjacent fragments are mixed and extended by complementary annealing of the overlapping region. Finally, the full-length gene is amplified using outer primers.

[0011] Preferably, the host bacterium is *Escherichia coli*, and the rare codons of the host bacterium are at least one of the following: arginine (AGG / AGA / CGA / CGG), leucine (CTT / CTC / CTA / TTA), proline (CCC), isoleucine (ATA), valine (GTA), glutamic acid (GAG), and glycine (GGG). The frequently used synonymous codons of the host bacterium correspond to arginine (CGT / CGC), leucine (CTG), proline (CCG), isoleucine (ATT), valine (GTG), glutamic acid (GAA), and glycine (GGC).

[0012] Preferably, the number of segments is 10, and the length of each segment is 450–500 bp.

[0013] Preferably, the 10 fragments are fragment 1 (1–455 bp), fragment 2 (436–890 bp), fragment 3 (871–1325 bp), fragment 4 (1306–1760 bp), fragment 5 (1741–2195 bp), fragment 6 (2176–2630 bp), fragment 7 (2611–3065 bp), fragment 8 (3046–3500 bp), fragment 9 (3481–3935 bp), and fragment 10 (3916–4401 bp). The nucleotide sequences of the PCR primers corresponding to fragments 1–10 are shown in SEQ ID NO.2–SEQ ID NO.21, respectively.

[0014] 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, the optimized design of this invention targets the unique Gly-XY repeat structure characteristic of full-length human type III collagen. Human type III collagen sequences contain numerous repeating Gly-Pro-X or Gly-X-Pro tripeptide structures, with amino acids such as glycine (Gly), proline (Pro), and arginine (Arg) accounting for a high proportion of the sequence. Therefore, this invention does not uniformly replace all rare codons during codon optimization, but rather prioritizes host-preferred codon optimization targeting rare codons containing these high-frequency amino acids. For example, codons rare in E. coli (such as AGA / AGG for arginine) in the original sequence are replaced with commonly used synonymous codons in E. coli (such as CGT / CGC). After optimization, the gene length is approximately 4401 bp, while maintaining the same amino acid sequence. This targeted optimization, based on the repetitive structural characteristics of collagen, improves translation efficiency across the entire multi-repetitive sequence, thereby enhancing the expression level of full-length collagen. The recombinant full-length human type III collagen Escherichia coli PE2586-hCo1 strain was deposited on January 28, 2026, at the China Center for Type Culture Collection (CCTCCNO: M2026274), located at Wuhan University, China.

[0015] Secondly, the codon optimization in this invention also considers the stability of ribosome movement during the translation of long sequences. The COL3A1 coding sequence is approximately 4401 bp long, much longer than typical bacterial protein sequences. During translation, ribosomes are prone to stalling or premature termination due to clusters of rare codons. This invention focuses on reducing the clustered distribution of consecutive rare codons in the sequence during optimization, making the translation process more continuous and stable, thereby improving the overall translation success rate of long protein sequences.

[0016] Third, this invention combines codon design with expression strategies. Instead of pursuing soluble expression, this invention aims to improve overall translation efficiency, enabling rapid protein accumulation within cells and the formation of stable inclusion body structures. For highly repetitive and complexly folded proteins like full-length human type III collagen, inclusion body expression can avoid intracellular degradation and achieve higher expression levels. Further centrifugation allows for precipitation and separation from bacterial lysates, containing a relatively pure target protein. Subsequent in vitro refolding processes ensure that the recombinant collagen within the inclusion bodies is obtained in a soluble, native conformation, laying the foundation for downstream functional studies and applications. Therefore, the codon optimization strategy of this invention is actually aligned with the overall technical approach of "inclusion body expression plus in vitro refolding," rather than simply optimizing expression efficiency.

[0017] Fourth, this invention utilizes an innovative segmented synthesis and overlap extension PCR splicing strategy to break down the challenging synthesis of the 4.4 kb full-length human type III collagen gene into multiple small fragments that are easy to synthesize and manipulate. Each adjacent fragment is designed with a 15–20 bp overlapping sequence region, allowing for seamless assembly of multiple fragments without the introduction of restriction sites. This precisely designed overlapping sequence region ensures that each synthesized fragment is sequentially connected, facilitating subsequent splicing and ensuring the accuracy and efficiency of the splicing process. It is particularly suitable for de novo synthesis of long genes. Attached Figure Description

[0018] Figure 1 The image shown is an SDS-PAGE gel image of recombinant full-length human collagen as described in this invention. Lane 1 represents the whole-cell control band without IPTG induction; Lane 2 represents the whole-cell sample band induced by IPTG; Lane 3 represents the supernatant sample band of IPTG-induced bacterial cells after ultrasonic lysis and centrifugation; Lane 4 represents the supernatant sample band of IPTG-induced bacterial cells after ultrasonic lysis and centrifugation, after resuspending in 1% Triton X-100 and centrifuging; Lane 5 represents the supernatant sample (column-loaded sample) of IPTG-induced bacterial cells after ultrasonic lysis and centrifugation, after resuspending in 8 mol / L urea and centrifuging; Lane 6 represents the first breakthrough peak band that was not adsorbed by the packing column after induction and eluted directly with the mobile phase; Lane 7 represents the second breakthrough peak band that was not adsorbed by the packing column after induction and eluted directly with the mobile phase; Lane 8 represents the 100 mmol / L imidazole elution fraction; Lane 9 represents the 200 mmol / L imidazole elution fraction: Lane 10 represents the Ni-removed fraction from EDTA buffer. 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] Example 1: Codon Optimization Design The wild-type full-length human type III collagen gene selected in this invention (derived from the cDNA of the human COL3A1 gene, NCBI ReferenceNM_000090.4) is 4401 bp in length. Due to its long sequence, the full-length human type III collagen gene is almost impossible to fold correctly in the host bacterial expression system, resulting in insoluble inclusion bodies. In addition, the wild-type full-length human type III collagen sequence contains a large number of repetitive Gly-Pro-X or Gly-X-Pro tripeptide structures, in which amino acids such as glycine (Gly), proline (Pro), arginine (Arg), leucine (Leu), isoleucine (Ile), valine (Val), and glutamic acid (Glu) account for a high proportion. The preferred codon types of these amino acids may differ between human and host bacteria. Therefore, during translation, the concentration of rare human codons can easily lead to ribosome arrest or premature termination in the host bacteria, resulting in low expression efficiency, low protein yield, and difficulty in achieving industrial production. Therefore, in this embodiment, at least one rare codon of glycine (Gly), proline (Pro), and / or arginine (Arg) corresponding to the Gly-Pro-X or Gly-X-Pro tripeptide structure in the wild-type full-length human type III collagen gene sequence is replaced with a synonym codon frequently used by the host bacterium to design and form an improved full-length human type III collagen gene. The host bacterium selected in this embodiment is *Escherichia coli*, therefore, it is necessary to preferentially replace the rare codons in *E. coli* encoding the tripeptide structure Gly-Pro-X or Gly-X-Pro with commonly used synonym codons in *E. coli*. However, it should be understood that the rare codons may differ for different expression host bacteria, and those skilled in the art can select rare codons that are mutated to fit the host bacterium species based on the sequence of the protein to be expressed and the type of host bacterium.In this embodiment, the optimization principles for the host bacterium *Escherichia coli* are shown in Table 1. In the wild-type full-length human type III collagen gene sequence, the commonly used codons encoding arginine are AGA / AGG / CGA / CG, but in *E. coli*, the high-frequency codons encoding arginine are usually CGT / CGC. In the wild-type full-length human type III collagen gene sequence, the commonly used codons encoding leucine are CTT / CTC / CTA / The common codon for leucine in wild-type full-length human type III collagen is TTA, but the high-frequency codon for leucine in E. coli is usually CTG; the common codon for proline in wild-type full-length human type III collagen gene sequence is CCC, but the high-frequency codon for proline in E. coli is usually CCG; the common codon for isoleucine in wild-type full-length human type III collagen gene sequence is ATA, but the high-frequency codon for isoleucine in E. coli is usually ATT; the common codon for valine in wild-type full-length human type III collagen gene sequence is GTA, but the high-frequency codon for valine in E. coli is GTA. The high-frequency codon is typically GTG; in the wild-type full-length human type III collagen gene sequence, the commonly used codon encoding glutamate is GAG, but in *E. coli*, the high-frequency codon encoding glutamate is typically GAA; in the wild-type full-length human type III collagen gene sequence, the commonly used codon encoding glycine is GGG, but in *E. coli*, the high-frequency codon encoding glycine is typically GGC. By replacing one or more of the above rare codon sites with high-frequency codons, and more preferably replacing all of the above sites, the translation efficiency of full-length human type III collagen in *E. coli* can be greatly improved. It should be understood that in codon optimization, this invention does not uniformly replace all rare codons, but rather prioritizes host-preferred codon optimization targeting these high-frequency amino acids. The optimized gene length is approximately 4401 bp, while maintaining the amino acid sequence unchanged. This targeted optimization based on the repetitive structure characteristics of collagen results in an overall improvement in translation efficiency throughout the multi-repetitive sequence, thereby contributing to increased expression levels of full-length type III collagen.

[0023] Table 1. Optimization Principles (Human Codons → Preferred Codons for E. coli) Example 2: Synthesis of an improved full-length human type III collagen gene Because this embodiment involves a large number of mutation sites, and the gene synthesis length cannot be too long (otherwise, the synthesis efficiency would be too low), this embodiment adopts a segmented synthesis method for the improved full-length human type III collagen gene (4000+ bp in length), which is then assembled using primers. In this embodiment, the designed improved full-length human type III collagen gene is divided into 6-12 segments, with each adjacent segment designed to have a 15-20 bp overlapping sequence region. Specifically, in this embodiment, the 4401 bp sequence of the improved full-length human type III collagen gene is divided into 10 segments, each 450–500 bp in length. Segment 1 (1–455 bp), segment 2 (436–890 bp), segment 3 (871–1325 bp), segment 4 (1306–1760 bp), segment 5 (1741–2195 bp), segment 6 (2176–2630 bp), segment 7 (2611–3065 bp), segment 8 (3046–3500 bp), segment 9 (3481–3935 bp), and segment 10 (3916–4401 bp) share 15–20 bp between any two adjacent segments. The overlapping regions of fragments are designed to allow for seamless assembly of multiple fragments without the need for restriction sites. For example, fragments 1 and 2 each contain overlapping sequences of 436–455 bp, fragments 2 and 3 each contain overlapping sequences of 871–890 bp, and so on. This precisely designed overlapping region ensures that the synthesized fragments are sequentially contiguous, facilitating subsequent splicing and ensuring the accuracy and efficiency of the splicing process. It is particularly suitable for de novo synthesis of long genes. Fragments 1–10 are then synthesized sequentially using artificial synthesis methods. Example 3: Primer Design Since each adjacent fragment includes a 15–20 bp overlapping sequence region, in this embodiment, specific primer sequences can be designed for the overlapping region of each fragment for fusion splicing between fragments. The primers are located at both ends of each fragment and are 19–22 nt in length. The primers are complementary to the first and last codons of the modified full-length human type III collagen gene fragments 1–10. Some primers may include synonymous codon mutation sites that are frequently used in E. coli, as shown in Table 1. The primer sequences between fragments 1–10 are shown in Table 2.

[0024] Table 2 Primer sequences between fragments 1 to 10 Example 4: Fragment splicing The splicing strategy in this embodiment is implemented using the overlap extension PCR (OE-PCR) method, that is: each fragment is amplified separately using primers with the overlapping sequence region, then adjacent fragments are mixed and extended by complementary annealing of the overlapping region, and finally the full-length gene is amplified using the outer primer.

[0025] Specifically, in this embodiment, the first round of PCR uses the artificially synthesized improved human type III collagen gene fragments 1 to 10 as templates and SEQ ID NO.2 to SEQ ID NO.21 as primers to amplify fragments 1 to 10 respectively, and obtains the amplified fragments 1 to 10 by gel recovery.

[0026] In the second round of PCR, fragment 1 and fragment 2 were combined and used as a template for amplification. SEQ NO.2F and SEQ NO.3R were used as primers for amplification and fragment 12 was obtained by gel recovery. Fragments 34, 56, 78 and 910 were obtained sequentially using the same method.

[0027] In the third round of PCR, fragments 12 and 34 were combined and used as amplification templates. SEQ NO.2F and SEQ NO.5R were used as primers, and fragments 12 and 34 were obtained by gel recovery. Fragment 56 and 78 could be obtained by the same method.

[0028] In the fourth round of PCR, the amplified fragments 1234 and 5678 were combined as amplification templates, and SEQ NO.2F and SEQ NO.9R were used as primers. The amplification was performed and the fragments 1234 and 5678 were recovered by gel.

[0029] In the fifth round of PCR, fragments 12345678 and 910 were merged and used as a template. SEQ NO. 2F and SEQ NO. 11R were used as primers. Amplification was performed and the sample was recovered via gel electrophoresis, ultimately yielding a full-length recombinant human type III collagen nucleic acid molecule of approximately 4.4 kb, as shown in SEQ NO. 1. This recombinant human type III collagen nucleic acid molecule successfully incorporated the synonymous codon mutation sites frequently used in *E. coli*, as shown in Table 1.

[0030] It should be understood that the splicing order of adjacent DNA fragments in this invention is only a preferred example. Those skilled in the art can also splice adjacent fragments sequentially using other combinations. The splicing order shown in this embodiment should not be construed as a limitation on the scope of protection of the claims. Furthermore, the host bacterium chosen for this invention is *Escherichia coli*, and the rare codons and frequently used synonymous codons are also compatible with *E. coli*. Those skilled in the art can select other corresponding rare codons and frequently used synonymous codons according to different host bacterium species.

[0031] Example 5: Construction of expression vector Using conventional molecular cloning techniques, the recombinant full-length human type III collagen nucleic acid molecule obtained by splicing was cloned into a high-efficiency expression vector in a prokaryotic expression system, thereby constructing the recombinant expression vector of the present invention. For example, in this embodiment, the pET series vector (such as pET-30a(+) / pET-28a(+) vectors with a T7 promoter) was selected for the recombinant expression vector. The recombinant human type III collagen nucleic acid molecule was inserted into the multiple cloning site of the pET series vector, and a His tag was attached downstream for purification. After the constructed recombinant expression vector was confirmed to have the correct sequence by sequencing, it was transformed into Escherichia coli BL21(DE3). The recombinant human type III collagen Escherichia coli (Escherichia coli) PE2586-hCo1 strain was deposited at the China Center for Type Culture Collection on January 28, 2026, with accession number CCTCCNO: M2026274, at Wuhan University, China. It should be understood that the above expression vectors and vector bacteria can be selected according to actual needs, and their specific models and strains should not be construed as limiting the scope of protection of the claims.

[0032] Example 6: Expression in Escherichia coli The recombinant strain was grown in a suitable culture medium and the target protein was expressed using IPTG. Due to codon optimization, the recombinant full-length human type III collagen gene was efficiently transcribed and translated in E. coli, resulting in a significant increase in protein expression. Figure 1 The image shown is an SDS-PAGE gel image of the recombinant full-length human type III collagen described in this invention. Lane 1 represents the uninduced whole-cell control band; Lane 2 represents the IPTG-induced whole-cell sample band; Lane 3 represents the supernatant sample band of IPTG-induced bacterial cells after ultrasonic lysis and centrifugation; Lane 4 represents the supernatant sample band of the precipitate after IPTG-induced bacterial cells after ultrasonic lysis and centrifugation, resuspended in 1% Triton X-100, and centrifuged; Lane 5 represents the supernatant sample (column-loaded sample) of the precipitate after IPTG-induced bacterial cells after ultrasonic lysis and centrifugation, resuspended in 8 mol / L urea, and centrifuged; Lane 6 represents the first breakthrough peak band, which was not adsorbed by the packing column after induction and eluted directly with the mobile phase; Lane 7 represents the second breakthrough peak band, which was not adsorbed by the packing column after induction and eluted directly with the mobile phase; Lane 8 represents the 100 mmol / L imidazole elution fraction; Lane 9 represents the 200... mmol / L imidazole elution fraction: Lane 10 represents the Ni-removed fraction from EDTA buffer.

[0033] The SDS-PAGE analysis showed that, without IPTG induction, the host bacteria did not exhibit a strong band of the target protein (Lane 1), demonstrating that the expression of the target protein was strictly controlled by the IPTG inducer. After IPTG induction, as indicated by the red arrows, a significant target protein (recombinant full-length human type III collagen) appeared at 140 kDa, constituting the majority of the total bacterial protein (Lane 2), indicating successful induction. Because recombinant full-length human type III collagen is a high-molecular-weight structural protein, the induced protein was almost entirely present in the bacterial cells as inclusion bodies (Lane 5, 4), rather than as a soluble protein in the cytoplasm (Lane 3). The inclusion bodies, containing a relatively pure target protein, could be separated from the bacterial lysate by centrifugation. The binding efficiency of the nickel column to the target protein was examined (Lane 6, 7). Only very weak bands of the target protein were observed in both lanes, indicating good binding ability of the nickel column and no loss of the target protein. Elution of the His-tagged protein on the column with different concentrations of competing reagents (100 mmol / L / 200 mmol / L imidazole) showed that the low concentration of 100 mmol / L imidazole was used to elute weakly bound impurities and some of the target protein (Lane 8), while the high concentration of 200 mmol / L imidazole completely eluted the target protein with very few impurity bands, proving that the nickel column achieved the expected purification effect (Lane 9). Further analysis using EDTA to chelate nickel ions (Ni²⁺)... + The column material was detached, thus eluting proteins with abnormally strong binding. The results showed that the main target protein was not present, indicating that the binding force between the target protein and the column was moderate and there were no non-specific interactions.

[0034] Example 7: Inclusion body dissolution and in vitro refolding Since the recombinant full-length human type III collagen expressed in this invention exists in bacterial cells as inclusion bodies, the dissolution and refolding of these inclusion bodies are crucial for restoring the native conformation and biological function of the recombinant full-length human type III collagen. In this embodiment, the isolated inclusion body proteins were thoroughly denatured and dissolved under 8 M urea or 6 M guanidine hydrochloride conditions, and then the denaturing agent was removed by stepwise dialysis or dilution. A molecular chaperone system was added to the refolding buffer to promote correct folding, such as adding an appropriate amount of GroEL / GroES molecular chaperone complex and ATP, or adding protein oxidases / isomerases or other folding aids to improve the refolding yield. Glycerol, L-Arginine, or other chemical chaperones can also be added to the refolding system to stabilize the intermediate protein structure and prevent re-aggregation. In this embodiment, inclusion body proteins were replaced with a refolding buffer containing an appropriate amount of oxidized glutathione / reduced glutathione system via gradient dialysis to promote disulfide bond formation. After a suitable refolding time, the target collagen obtained correct folding and was able to specifically bind to the collagen-binding reagent, demonstrating that its functional domains had recovered their normal conformation. The entire refolding process resulted in the recombinant full-length human type III collagen in the inclusion bodies being obtained in a soluble, native conformation, laying the foundation for downstream functional studies and applications.

[0035] In summary, this invention achieves efficient expression and renaturation of a full-length human type III collagen gene in prokaryotic expression systems, particularly in *E. coli*. The core of this invention lies in codon optimization of the gene to *E. coli*-preferred codons, which improves translation efficiency. The full-length gene was constructed using segmented synthesis and overlapping sequence splicing methods, allowing for seamless assembly of multiple fragments without the introduction of restriction sites. Mature inclusion bodies are generated during the expression stage, and bioactive collagen is obtained after in vitro chaperone-assisted renaturation. This has broad application prospects for the development of biomedical materials, such as the preparation of tissue engineering scaffolds, wound dressings, or cosmetic preparations. This technical solution provides a feasible route for preparing high-quality human macromolecular collagen in prokaryotic systems. With similar methods and appropriate optimization, it can also be applied to the preparation of other high-molecular-weight proteins that are difficult to express in *E. coli*.

[0036] 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.

[0037] 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 nucleic acid molecule encoding recombinant full-length human type III collagen, characterized in that, The nucleic acid molecule is designed with codon optimization, replacing at least one glycine, proline, arginine, leucine, isoleucine, valine and / or glutamic acid codons corresponding to the Gly-Pro-X or Gly-X-Pro tripeptide structure in the wild-type full-length human type III collagen gene sequence with synonyms that are frequently used by the host bacteria, while keeping the amino acid length and sequence unchanged.

2. The nucleic acid molecule encoding recombinant full-length human type III collagen according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

1.

3. A recombinant expression vector containing the nucleic acid molecule as described in claim 1 or 2.

4. A recombinant expression strain, characterized in that, Includes the recombinant expression vector as described in claim 3.

5. The recombinant expression strain according to claim 4, characterized in that, The host bacteria of the recombinant expression strain include at least one of Escherichia coli, Bacillus subtilis, and lactic acid bacteria.

6. The use of the recombinant expression strain according to claim 4 or 5 in the fields of tissue engineering and cosmetic surgery.

7. A method for synthesizing recombinant full-length human type III collagen nucleic acid molecules, characterized in that, Includes the following steps: S1. Codon optimization design: Replace at least one glycine, proline, arginine, leucine, isoleucine, valine and / or glutamic acid codon corresponding to the Gly-Pro-X or Gly-X-Pro tripeptide structure in the wild-type full-length human type III collagen gene sequence with a synonym codon frequently used by the host bacteria to design an improved full-length human type III collagen gene. S2. Synthesis of improved full-length human type III collagen gene: The improved human type III collagen gene is divided into 6-12 fragments, and each adjacent fragment is designed with a 15-20 bp overlapping sequence region. The above 6-12 fragments are synthesized sequentially by artificial synthesis. S3. Primer design: Design specific primer sequences for the overlapping sequence regions of each of the fragments; S4. Fragment splicing: Each fragment is amplified separately using primers containing the overlapping sequence region. Then, adjacent fragments are mixed and extended by complementary annealing of the overlapping region. Finally, the full-length gene is amplified using outer primers.

8. The method for synthesizing recombinant full-length human type III collagen nucleic acid molecules according to claim 7, characterized in that, The host bacterium is *Escherichia coli*. The rare codons of the host bacterium are at least one of the following: arginine (AGG / AGA / CGA / CGG), leucine (CTT / CTC / CTA / TTA), proline (CCC), isoleucine (ATA), valine (GTA), glutamic acid (GAG), and glycine (GGG). The frequently used synonymous codons of the host bacterium correspond to arginine (CGT / CGC), leucine (CTG), proline (CCG), isoleucine (ATT), valine (GTG), glutamic acid (GAA), and glycine (GGC).

9. The method for synthesizing recombinant full-length human type III collagen nucleic acid molecules according to claim 8, characterized in that, The number of segments is 10, and the length of each segment is 450–500 bp.

10. The method for synthesizing recombinant full-length human type III collagen nucleic acid molecules according to claim 9, characterized in that, The 10 fragments are fragment 1 (1–455 bp), fragment 2 (436–890 bp), fragment 3 (871–1325 bp), fragment 4 (1306–1760 bp), fragment 5 (1741–2195 bp), fragment 6 (2176–2630 bp), fragment 7 (2611–3065 bp), fragment 8 (3046–3500 bp), fragment 9 (3481–3935 bp), and fragment 10 (3916–4401 bp). The nucleotide sequences of the PCR primers corresponding to fragments 1–10 are shown in SEQ ID NO.2–SEQ ID NO.21, respectively.

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