A method for reconstituting collagen triple helix folding and stabilization

Abstract

The application belongs to the technical field of protein engineering, and particularly relates to a recombinant collagen triple helix folding and stabilizing method, and a technology for assisting efficient expression of human recombinant collagen in Escherichia coli to form a stable triple helix structure. The technology focuses on solving the key technical problems of low folding efficiency and unstable triple helix structure of the recombinant collagen in the Escherichia coli expression system, and significantly improves the yield of functional recombinant collagen. The application has important application value in the fields of biomedical materials, tissue engineering, beauty and skin care, and provides an efficient and economical technical path for industrial production of recombinant collagen.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering technology in the biotechnology field, specifically involving a technology that efficiently assists human recombinant collagen expressed in Escherichia coli to fold correctly and form a stable triple helix structure by optimizing and customizing a prokaryotic molecular chaperone system. Background Technology

[0002] Collagen, as one of the most important structural proteins in the human body, plays a crucial role in maintaining tissue integrity, promoting wound healing, and facilitating cell adhesion. Its unique triple helix structure is fundamental to its biological functions, making collagen a promising candidate for applications in biomedical materials, skincare, and functional foods. Traditional animal-derived collagen carries risks such as viral contamination and immunogenicity, while recombinant collagen technology offers a viable solution to these problems.

[0003] Currently, the production of recombinant collagen mainly utilizes gene engineering expression systems, including prokaryotic Escherichia coli expression systems, eukaryotic yeast expression systems, and insect cell expression systems. Among these, the E. coli expression system has become the preferred method for industrial production due to its advantages such as low culture cost, ease of operation, and high expression levels. However, the E. coli system has significant drawbacks when expressing complex human collagen: the expression product easily forms insoluble inclusion bodies, requiring complex refolding processes; the lack of a eukaryotic folding environment makes it difficult for recombinant collagen to form a stable triple helix structure; and it cannot complete crucial post-translational modifications. A correct triple helix structure is fundamental to ensuring collagen activity and function, and is a key technological barrier in recombinant humanized collagen production, thus directly affecting the bioactivity of collagen.

[0004] Escherichia coli possesses its own molecular chaperone system, including Trigger Factor, DnaK / DnaJ, and GroES / GroEL, which play crucial roles in protein folding. Trigger Factor, a ribosome-associated chaperone, primarily participates in the early folding of nascent polypeptide chains; DnaK / DnaJ reduces the aggregation of misfolded proteins and promotes protein hydrolysis, stabilizing unfolded proteins; while GroES / GroEL provides highly coordinated and symmetrical allosteric assistance in protein folding. However, endogenous molecular chaperone systems are often insufficient in assisting the folding of highly expressed heterologous proteins, especially structurally complex proteins like collagen, leading to misfolding and aggregation of the expression products. Therefore, developing a molecular chaperone-assisted technology that can efficiently guide recombinant collagen to form the correct triple helix structure has become a critical problem that urgently needs to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention aims to provide a system that, through customized and optimized prokaryotic molecular chaperone systems, particularly utilizing E. coli's own Trigger Factor (TF), DnaK / DnaJ chaperone protein pairs, and GroES / GroEL chaperone protein pairs, efficiently assists human recombinant collagen expressed in E. coli to correctly fold from its denatured state and form a biologically active, stable triple helix structure. This solves the problems of low refolding efficiency of recombinant collagen, difficulty in forming triple helix structures, and low yield of functional products.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] On the one hand, this application provides a method for efficient triple helix folding and stabilization of recombinant collagen, comprising:

[0008] 1) Preparation and preliminary treatment of recombinant collagen; 2) Trigger Factor and DnaK / DnaJ were added immediately at the beginning of refolding, and the reaction was carried out at 3-5℃ for 0.5-2 hours; GroES / GroEL and ATP were added when the urea concentration dropped to 0.2-0.6M;

[0009] Furthermore, ATP is replenished every 3-5 hours to maintain molecular chaperone activity, wherein the ATP is 0.5-1.5 mM.

[0010] Further, the concentration of the Trigger Factor is 0.05-0.3 mg / mL, preferably 0.06-0.2, more preferably 0.08-0.15; the molar ratio of DnaK / DnaJ to target protein is 0.5-2:0.5-2, preferably 0.8-1.2:0.8-1.2; the molar ratio of GroES / GroEL is 0.8-1.2:0.8-1.2; the molar ratio of DnaK:DnaJ is 0.9-1.1:0.1-0.3; and the molar ratio of Trigger Factor:DnaK / DnaJ:GroEL is 1.8-2.2:0.8-1.2:0.8-1.2.

[0011] Furthermore, when the urea concentration drops to 2-6 M, GroES / GroEL and 1-4 mM ATP are added.

[0012] Furthermore, the preparation and preliminary processing of human recombinant collagen includes recombinant expression and inclusion body induction, inclusion body extraction and purification, and inclusion body denaturation and dissolution steps.

[0013] Furthermore, the recombinant expression and inclusion body induction include using BL21(DE3) Escherichia coli as the expression host and cloning the codon-optimized human type III collagen (COL3A1) gene into the pET-28a(+) vector.

[0014] On the one hand, this application provides a functional recombinant collagen prepared by the above-mentioned preparation method.

[0015] A pioneering three-molecule chaperone synergistic refolding system: For the first time, three molecular chaperones of E. coli, namely Trigger Factor, DnaK / DnaJ and GroES / GroEL, are used in combination in a specific order and ratio, which significantly improves the refolding efficiency of collagen.

[0016] This application addresses key technical challenges commonly encountered in E. coli expression systems, such as low folding efficiency and unstable triple-helix structure, significantly improving the yield of functional recombinant collagen. This invention has significant application value in biomedical materials, tissue engineering, and cosmetic skincare, providing an efficient and economical technical pathway for the industrial production of recombinant collagen. Attached Figure Description

[0017] Figure 1 The yield of refolded proteins in different combinations of molecular chaperones.

[0018] Figure 2 Circular dichroism (CD) confirmed the presence of the characteristic triple helix structure. Detailed Implementation

[0019] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0020] Example

[0021] A method for assisting the formation of recombinant collagen triple helix structure using a customized molecular chaperone system

[0022] 1. Preparation and preliminary processing of recombinant human collagen

[0023] 1) Recombinant expression and inclusion body induction

[0024] Using BL21(DE3) *E. coli* as the expression host, the codon-optimized human type III collagen (COL3A1) gene was cloned into the pET-28a(+) vector. During the seed culture stage, LB medium containing 50 μg / mL kanamycin was used, and the culture was carried out at 37°C with shaking at 200 rpm until OD600 ≈ 0.6. Subsequently, the culture was transferred to TB medium (containing 0.5% glucose), and when OD600 reached 0.8, 0.5 mM IPTG was added, followed by induction at 30–37°C for 16 hours. This temperature induction condition effectively promoted inclusion body formation.

[0025] 2) Inclusion body extraction and purification

[0026] After bacterial cell collection, inclusion bodies were purified using a three-step washing method. First, the cells were washed twice with PBS (pH 7.4) to remove residual culture medium. Then, they were treated three times with a washing buffer containing 2 M urea and 1% Triton X-100 to effectively remove membrane protein and nucleic acid contamination. Finally, the cells were washed twice with a low-salt buffer (20 mM Tris-HCl, 1 mM EDTA, pH 8.0) to ensure the removal of residual detergent. The entire process was performed at 4°C to maintain protein stability.

[0027] 3) Inclusion body denaturation and dissolution

[0028] Inclusion bodies were dissolved using 6 M guanidine hydrochloride or 8 M urea denaturing buffer (20 mM Tris-HCl, pH 8.0, containing 5 mM MdTT reducing agent). Dissolution was carried out at 25°C with stirring for 2–4 hours until complete dissolution (or overnight at 4°C), followed by centrifugation at 15,000 × g for 30 min to remove insoluble matter. The denatured and dissolved protein was obtained after filtration through a 0.22 μm filter. Protein concentration was determined using the BCA method and adjusted to 0.5 mg / mL–1 mg / mL to prepare for subsequent renaturation experiments.

[0029] 2. Methods for obtaining molecular chaperones

[0030] 1) Preparation of Trigger Factor (TF): The *E. coli* tig gene was cloned into the pET-28a(+) vector (with an N-terminal His-tag) and transformed into BL21(DE3) competent cells. Expression was induced using TB medium (containing 50 μg / mL kanamycin) at 37°C until OD600≈0.6, then 0.5 mM IPTG was added, and the cells were induced at 16°C for 12-16 hours to reduce inclusion body formation. Purification steps included: cell sonication (lysis buffer: 20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0); centrifugation of the lysis buffer followed by nickel column affinity chromatography (binding buffer containing 20 mM imidazole, elution buffer containing 250 mM imidazole); the eluted fraction was desalted using a PD-10 column and replaced with storage buffer (20 mM Tris-HCl, 100 mM KCl, 10% glycerol, pH 7.5). The final product must meet the following requirements: concentration ≥ 5 mg / mL, purity > 95% as determined by SDS-PAGE, and stored at -80℃ after aliquoting to avoid repeated freeze-thaw cycles.

[0031] 2) DnaK / DnaJ preparation: The expression system uses pKJE7 plasmid (containing dnak and dnaj genes, of which DnaK carries a His-tag), which is transformed into BL21(DE3) and cultured in LB medium (containing 34 μg / mL chloramphenicol) at 30℃ until OD600≈0.6, and then 0.5 mM IPTG is added for induction for 6 hours (to avoid DnaK aggregation caused by high temperature).

[0032] The purification process was performed in steps: DnaK was obtained by nickel column affinity chromatography and concentrated to 10-15 mg / mL by ultrafiltration; DnaJ was separated by ion exchange chromatography (Q column), with DnaJ flowing through 50 mM NaCl and further purified by Superdex 200 gel filtration. The complexes were reconstituted and mixed at a molar ratio of 1:0.2 (DnaK:DnaJ) and incubated at 4°C for 1 hour. The working concentrations of the final products were 10 μM DnaK and 2 μM DnaJ, respectively, with 2 mM ATP added to the buffer to maintain activity periodically.

[0033] 3) GroES / GroEL Preparation: The expression system used pGro7 plasmid (containing groES and groEL genes, with GroEL carrying a His-tag). After transformation into BL21(DE3), the plasmid was cultured in TB medium (containing 34 μg / mL chloramphenicol) at 37°C until OD600≈0.6. 0.5 mg / mL L-arabinose was added to induce the pBAD promoter, and the mixture was induced at 25°C for 8 hours to promote complex formation. Purification involved first capturing GroEL-His using a nickel column, and then directly obtaining the GroES / GroEL complex via elution. Free GroES was removed by Superose 6 gel filtration chromatography to ensure the complex was present in a strict 1:1 ratio (GroES:GroEL). The final product was concentrated by ultrafiltration to 4-8 mg / mL GroEL (working concentration 4-8 μM), with the GroES concentration approximately half that of GroEL. Before use, 2 mM ATP was added to the buffer, and 1 mM was added every 4 hours to maintain the folding activity of GroEL.

[0034] 3. Construction and optimization of customized molecular chaperone systems

[0035] 1) Optimization of the refolding process

[0036] The refolding process employs a dilution method, where denatured proteins are slowly added at a 1:10 ratio to a pre-cooled refolding buffer containing 50 mM Tris-HCl, 150 mM NaCl, and 5 mM GSH / GSSG, reducing the final urea concentration to below 1 M. At this denaturant concentration, collagen is in a partially unfolded state, making it easier for early-acting molecular chaperones to capture it.

[0037] During the renaturation process, we designed seven molecular chaperone combination experiments: 1) using TF alone; 2) using DnaK / DnaJ alone; 3) using GroES / GroEL alone; 4) TF + DnaK / DnaJ; 5) TF + GroES / GroEL; 6) DnaK / DnaJ + GroES / GroEL; 7) TF + DnaK / DnaJ + GroES / GroEL. All experimental groups used gradient dialysis, with the urea concentration gradually decreasing from 1M to 0M, each step lasting 12 hours, and the temperature controlled at 4℃.

[0038] Of particular note is that in the triple molecular chaperone combination group (TF+DnaK / DnaJ+GroES / GroEL), we optimized the order and timing of addition: TF (0.1 mg / mL) and DnaK / DnaJ (molar ratio to target protein 1:1) were added immediately at the start of renaturation, and the reaction was carried out at 4°C for 1 hour; when the urea concentration dropped to 0.4 M, GroES / GroEL (1:1 molar ratio) and 2 mM ATP were added, and the dialysis process was continued, with 1 mM ATP supplemented every 4 hours to maintain molecular chaperone activity.

[0039] Optimization of the Three-Molecular Chaperone Co-administration Strategy: In achieving the combined folding assistance of three molecular chaperones—Trigger Factor (TF), DnaK / DnaJ, and GroES / GroEL—we focused on optimizing the order, timing, and ratio of their addition to maximize collagen refolding efficiency and yield. Based on theoretical analysis, TF, as a ribosome-binding chaperone, typically contacts the nascent peptide chain first and should be added early in the refolding process. Subsequently, DnaK / DnaJ (Hsp70 / DnaJ system) captures some folding intermediates and prevents aggregation, and finally, GroES / GroEL (Hsp60 system) assists in completing terminal folding. Therefore, we hypothesized that sequential stepwise addition (TF→DnaK / DnaJ→GroES / GroEL) is superior to simultaneous addition. Furthermore, to determine the optimal ratio of the three, we considered their physiological concentrations in cells and their mechanisms of action, speculating that a relatively higher TF dosage is more conducive to initial folding (the molar ratio of TF:DnaK:GroEL is approximately 2:1:1). To verify the above hypothesis, we designed a series of comparative experiments to optimize the order and proportion of addition:

[0040] a) Optimization of the addition order: Three different partner addition strategies were set: (A) all partners were added at the beginning of renaturation; (B) a "two-stage method" was used, i.e., TF was added at the beginning of renaturation, and DnaK / DnaJ and GroES / GroEL were added simultaneously after a certain delay; (C) a "three-stage method" was used, i.e., TF was added step by step in the order of TF→DnaK / DnaJ→GroES / GroEL. Strategy C is the preferred scheme of this invention: TF is added immediately at the beginning of renaturation, DnaK / DnaJ is added after a preset time, and GroES / GroEL is added after a further certain time. All experiments were performed under gradient dialysis renaturation at 4℃ to ensure comparability.

[0041] b) Optimization Experiment: After determining the optimal order, we further investigated the effects of the ratios of TF, DnaK / DnaJ, and GroES / GroEL on the folding effect. Using a target protein concentration of approximately 0.5 mg / mL as a baseline, we examined the effects of low, equal, and high TF dosages (TF to target protein molar ratios of 0.4:1, 2:1, and 4:1), as well as the effects of different ratios of DnaK / DnaJ and GroES / GroEL (TF:DnaK / DnaJ = 1:1, 1:0.5; TF:GroES / GroEL = 1:1, 1:0.5) on folding. Under each condition, other variables such as temperature, buffer composition, and dialysis rate remained constant, allowing for single-factor analysis of the ratios.

[0042] The above optimization experiments used the yield of correctly folded soluble collagen and refolding efficiency as evaluation indicators. Soluble proteins in the supernatant were analyzed by SDS-PAGE gel electrophoresis. The band intensity of the target protein under different conditions was compared, and the protein concentration was quantified using the BCA method to estimate the refolding yield. Simultaneously, the presence of a correct triple helix structure in the folded product was confirmed by limited enzymatic digestion and circular dichroism (CD) spectroscopy to assess the quality of the refolding effect.

[0043] Results Analysis: Experimental data showed that the order of addition of the three chaperones significantly affected the refolding effect. While simultaneous addition (all three chaperones added at once) improved the refolding yield to some extent, it was still insufficient compared to stepwise addition. The "two-stage method" (TF first, then the others) reduced early misaggregation to some extent, resulting in an increase in refolded products. However, the "three-stage method," adding TF → DnaK / DnaJ → GroES / GroEL in that order, significantly increased the yield of soluble target proteins compared to other strategies. Specifically, under optimal conditions (TF added at 0h, DnaK / DnaJ added 0.5h after the start of refolding, and GroES / GroEL added 1h after the start of refolding), the yield of correctly folded collagen reached approximately 75%, a significant improvement compared to ~15% without chaperones and ~60% with only simultaneous addition. Conversely, adding GroES / GroEL too early (synchronized with TF) reduced the refolding efficiency, possibly due to the limited effect of GroEL in the presence of high denaturant concentrations and premature ATP consumption. The above results corroborate that providing molecular chaperones in a sequential, phased manner better meets the needs of collagen folding: in the initial stage, TF works synergistically with DnaK / DnaJ to rapidly capture and stabilize nascent peptide chains, preventing irreversible aggregation; subsequently, GroES / GroEL are introduced in the mid-renaturation stage to take over the intermediate products under suitable conditions and promote their correct folding.

[0044] Meanwhile, experiments with different proportions showed that appropriately increasing the relative amount of TF improved refolding efficiency. When TF was added in a 2:1:1 molar ratio with DnaK / DnaJ and GroEL, the total amount of folded products and the correct folding ratio both reached their maximum. Further increasing the TF ratio to 4:1:1 did not significantly increase the yield, indicating a saturation point. Furthermore, optimization of the DnaK / DnaJ ratio showed that a DnaK:DnaJ ratio of 1:0.2 was optimal; excessive DnaJ might competitively inhibit the effect of DnaK. A GroES:GroEL ratio of 1:1 was optimal, ensuring synergistic GroES capping of each GroEL cavity. Under the optimized combination conditions, the yield of soluble collagen in the refolding solution increased several times compared to the unoptimized scheme, exhibiting clear and complete type III procollagen bands; while the amount of incorrectly folded proteins and macromolecular aggregates was significantly reduced. Therefore, by adjusting the order and ratio of the three molecular chaperones, this invention effectively guides the folding pathway of recombinant collagen, significantly improving the efficiency and yield of correct assembly of the collagen triple helix structure. This has not been reported in the prior art, demonstrating its innovation and superiority.

[0045] like Figure 1 As shown, the results are analyzed as follows: the single molecular chaperone groups TF only, DnaK / DnaJ only, and GroES / GroEL only showed weak monomeric bands (few soluble proteins); the bimolecular chaperone groups TF+DnaK / DnaJ, TF+GroES / GroEL, and DnaK / DnaJ+GroES / GroEL were better than the single molecular chaperone groups, but lower than TF+DnaK / DnaJ+GroES / GroEL.

[0046] Trimolecular chaperone combinations > TF + GroES / GroEL > TF + DnaK / DnaJ > Single chaperone combinations.

[0047] 4. Purification and Identification of Functional Recombinant Collagen

[0048] Subsequent purification: Functional recombinant collagen that has successfully folded and formed a triple helix structure is separated and purified by chromatography, removing incorrectly folded proteins, aggregates, residual molecular chaperones and other impurities.

[0049] (a) Ion Exchange Chromatography: Initial separation was achieved by utilizing the difference in isoelectric points between the target collagen and its chaperone proteins, selecting an appropriate ion exchange medium. The specific steps were as follows: the refolded protein solution was buffered to 20 mM MES (pH 6.0) with low ionic strength (NaCl ≤ 50 mM) via ultrafiltration or dialysis, and then loaded onto a pre-equilibrated SP column (column volume ~5 mL) at a flow rate of 1.0 mL / min. Unbound impurities and chaperone proteins (such as GroEL, DnaK, TF, etc., which are negatively charged at pH 6.0 and thus do not bind to the resin) were washed away during loading and subsequent low-salt washing (20 mM MES, 50 mM NaCl). Subsequently, the collagen bound to the resin was eluted using gradient elution (gradually increasing NaCl from 50 mM to 1 M, with a gradient volume of 20 column volumes). Experiments showed that the target recombinant collagen typically began elution at a NaCl concentration of approximately 0.3–0.5 M. Elution peaks were collected and each component was analyzed by SDS-PAGE. The results showed that the collagen subunit bands corresponding to the main elution peaks were clear, while a large number of characteristic bands of chaperone proteins such as DnaK and TF were detected in the flow-through component, proving that the cation exchange step effectively separated most of the chaperone proteins from the target protein.

[0050] (b) Gel filtration chromatography: After concentrating the crude collagen obtained by ion exchange elution, further purification was performed using gel filtration chromatography (also known as size exclusion chromatography). We used a Superose 6 extended column (10×300mm), and placed the sample in a buffer of 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, loaded onto the column, and separated at a flow rate of 0.5 mL / min. Due to size differences, the folded collagen triple helix (trimer) will elute at a specific volume, while any residual low molecular weight impurities and molecular chaperones will appear at a larger elution volume. Simultaneously, if a small amount of undissolved collagen aggregates or polymeric forms are present, their molecular weight is much higher than that of a single triple helix, and they will elute prematurely near the dead volume of the column, thus separating from the target product. The chromatogram of gel filtration typically shows two main peaks: the first small peak appears near the column void volume, containing trace amounts of high molecular weight aggregates; the second main peak corresponds to collagen. The main peak was collected and subjected to SDS-PAGE and silver staining analysis again. The results confirmed that its purity was >95% and that it did not contain bands of chaperone proteins such as DnaK and GroEL. After the above two-step chromatography, the recombinant collagen was highly purified, and the chaperone protein residues and other impurities from the refolding process were successfully removed.

[0051] Identification of the triple helix structure: by circular dichroism (CD) analysis (see...) Figure 2The recombinant collagen prepared showed a characteristic positive peak at 228 nm (molar ellipticity +3000 deg·cm). 2 ·dmol -1 The peak at 198 nm is negative (-5000 deg·cm). 2 ·dmol -1 ), and the spectrum of natural collagen Figure 1 The thermal denaturation test showed that its Tm value was 38±1℃, indicating that the triple helix structure has good stability.

[0052] This application pioneers a three-molecule chaperone synergistic refolding system, which for the first time combines three groups of molecular chaperones from E. coli—Trigger Factor, DnaK / DnaJ, and GroES / GroEL—in a specific order and ratio, significantly improving collagen refolding efficiency.

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing recombinant collagen triple helix folding and stabilization, characterized in that, The preparation method includes: 1) preparation and preliminary treatment of collagen, wherein the preparation and preliminary treatment of recombinant collagen includes recombinant expression and inclusion body induction, inclusion body extraction and purification, and inclusion body denaturation and dissolution steps, wherein inclusion body dissolution is performed using 8M urea denaturation buffer; 2) Trigger Factor and DnaK / DnaJ chaperone protein pair are added immediately at the start of renaturation, and the reaction is carried out at 3-5℃ for 0.5-2 hours; when the urea concentration drops to 0.2-0.6M, GroES / GroEL chaperone protein pair and ATP are added.

2. The preparation method according to claim 1, characterized in that, When the urea concentration drops to 0.2-0.6M, add GroES / GroEL chaperone protein pair and ATP, and continue the dialysis process. Supplement with 0.5-1.5 mM ATP every 3-5 hours to maintain molecular chaperone activity.

3. The preparation method according to claim 1, characterized in that, The recombinant expression and inclusion body induction involved using BL21(DE3) Escherichia coli as the expression host.

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