A method for constructing stable triple-helix recombinant collagen without hydroxyproline modification, its product and applications

Abstract

This invention relates to the fields of biomaterials and genetic engineering, providing a method for constructing a stable triple-helix recombinant collagen without hydroxyproline modification, as well as its products and applications. Based on the characteristic Gly-X-Y repeat sequence of collagen, this invention constructs a recombinant collagen that can form a stable triple-helix conformation even without hydroxyproline by regulating the amino acid composition and sequence arrangement at the X and Y positions. The recombinant collagen can form a stable triple-helix structure in aqueous solution, with a thermal denaturation temperature Tm ≥ 40 °C, and can self-assemble into a fibrous morphology under physiological conditions. Experimental results show that the recombinant collagen has good structural stability and biocompatibility. This invention provides a method for designing collagen that can construct a stable triple-helix structure without relying on hydroxyproline modification, which can be used to prepare tissue engineering scaffolds, skin repair materials, and medical biomaterials.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials and genetic engineering technology, specifically to a method for constructing a stable triple-helix recombinant collagen without hydroxyproline modification, its product, and its applications. Background Technology

[0002] Collagen is one of the most abundant structural proteins in mammals, widely found in connective tissues such as skin, bones, tendons, ligaments, cartilage, and blood vessels, playing a vital role in maintaining the mechanical strength and structural integrity of these tissues. A significant characteristic that distinguishes collagen from other proteins is that it consists of three polypeptide chains with a (Gly-XY)n repeating sequence intertwined to form a stable triple helix conformation, which further self-assembles into collagen fibers and higher-level structures.

[0003] The triple helix structure is fundamental to the structural and biological functions of collagen, and its stability directly affects the mechanical properties, structural integrity, and interactions with cells and other biomolecules. Studies have shown that the stability of the triple helix structure depends on specific amino acid composition and intramolecular interactions, with hydroxyproline considered to play a crucial role in the stability of the triple helix structure in natural collagen. However, in recombinant expression systems, due to the lack of a hydroxyproline modification mechanism, the obtained collagen often fails to form a stable triple helix structure, thus limiting its structural properties and application effects.

[0004] Currently, collagen is mainly obtained through animal tissue extraction and recombinant expression via genetic engineering. While animal-derived collagen has good bioactivity, it also presents challenges such as unstable sources, potential immunogenicity, and the risk of viral contamination. Recombinant collagen, on the other hand, offers advantages such as well-defined composition and high controllability. However, it still faces a key technological bottleneck in practical applications: the difficulty in constructing a stable triple helix structure without relying on hydroxyproline modification.

[0005] Furthermore, the stability of the triple helix structure of collagen not only affects its molecular-level structure but also directly determines whether it can further self-assemble into higher-order structures with fibrous morphology, which is a crucial foundation for its function as a biomaterial. Therefore, how to construct a stable triple helix structure under hydroxyproline-free conditions and achieve its further fibrous self-assembly has become a key technical problem that urgently needs to be solved in the field of recombinant collagen. Summary of the Invention

[0006] To address the problem that recombinant collagen in the prior art is difficult to form a stable triple helix structure under conditions without hydroxyproline modification, and is also difficult to further construct a fibrous morphology structure, this invention provides a method for constructing a stable triple helix structure of recombinant collagen, as well as its products and applications.

[0007] This invention, based on the characteristic Gly-XY repeat sequence of collagen, constructs a recombinant collagen that can form a stable triple helix conformation even without hydroxyproline by regulating the composition and arrangement of amino acids at the X and Y positions. The recombinant collagen exhibits high structural stability in aqueous solution, with a thermal denaturation temperature Tm ≥ 40 °C, and can further self-assemble under physiological conditions to form a fibrous structure.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a highly stable triple-helix recombinant collagen, wherein the highly stable triple-helix recombinant collagen comprises a polypeptide chain composed of Gly-XY repeating sequences, wherein:

[0010] ① The amino acid at position X is selected from one or more of Pro, Asp, and Glu;

[0011] ② The amino acid at the Y position is selected from one or more of Pro, Lys, and Arg;

[0012] And it satisfies the following structural characteristics:

[0013] (1) Contains no hydroxyproline;

[0014] (2) It can form a triple helix conformation in aqueous solution;

[0015] (3) Its thermal denaturation temperature Tm ≥ 40 ℃;

[0016] (4) It can self-assemble into a fibrous structure under physiological conditions.

[0017] Preferably, the amino acid sequence of the highly stable triple-helix recombinant collagen contains (Gly-XY). n Repeating units, where n is 10~30.

[0018] Preferably, the amino acid sequence of the highly stable triple helix recombinant collagen contains (Gly-Pro-Lys-Gly-Asp-Pro). n Structural unit.

[0019] Preferably, the amino acid sequence of the highly stable triple helix recombinant collagen is as shown in SEQ ID NO.1 or a variant thereof with the same function.

[0020] Preferably, the recombinant collagen with a highly stable triple helix structure exhibits a positive peak at ~225 nm and a negative peak at ~192 nm as measured by circular dichroism spectroscopy.

[0021] Preferably, the fibers formed by the self-assembly of the highly stable triple-helix recombinant collagen have a network structure morphology.

[0022] Secondly, the present invention provides a method for constructing the highly stable triple helix structure of recombinant collagen, characterized in that the composition and arrangement of amino acids at the X and Y positions in the Gly-XY repeat sequence are regulated to enable the recombinant collagen to form a highly stable triple helix structure under conditions without hydroxyproline.

[0023] Thirdly, the present invention provides a method for preparing recombinant collagen with the aforementioned highly stable triple helix structure, comprising the following steps:

[0024] (1) Construct the nucleotide sequence encoding the recombinant collagen;

[0025] (2) The nucleotide sequence is ligated into an expression vector and transformed into a host cell;

[0026] (3) Induced expression and purification to obtain the recombinant collagen with the highly stable triple helix structure.

[0027] Preferably, the expression vector includes pCold or pET.

[0028] Preferably, the host cell includes Escherichia coli or yeast.

[0029] Fourthly, the present invention provides the application of the highly stable triple helix structure of recombinant collagen in the preparation of biomaterials.

[0030] Preferably, the biomaterial includes tissue engineering scaffolds, skin repair materials, or medical biomaterials.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) This invention achieves the construction of a highly stable triple helix structure by regulating the Gly-XY repeat sequence without relying on hydroxyproline modification, providing a new structural regulation method for the formation of the triple helix conformation of recombinant collagen;

[0033] (2) The recombinant collagen of the present invention has high structural stability, can maintain the triple helix conformation in aqueous solution, and exhibits high thermal stability;

[0034] (3) The recombinant collagen described in this invention can self-assemble into a fibrous structure under physiological conditions, providing a structural basis for its application in biomaterials;

[0035] (4) The recombinant collagen described in this invention is obtained by recombinant expression and has the characteristics of clear composition and good batch consistency;

[0036] (5) The construction method described in this invention has certain universality and can be used to construct triple helix structures of different Gly-XY sequence systems;

[0037] (6) The recombinant collagen of the present invention has good water solubility and stability. Attached Figure Description

[0038] Figure 1 SDS-PAGE images of highly stable triple-helix recombinant collagen rSTH-Col prepared from different batches;

[0039] Figure 2 The image shows the Western blot (WB) of the highly stable triple helix recombinant collagen rSTH-Col; WT is the negative control, and 1, 2, and 3 are the supernatants after the expression of the three monoclonal strains with the highest expression levels.

[0040] Figure 3 Circular dichroism (a), thermal distortion (b), and thermal distortion first derivative (c) curves of highly stable triple helix recombinant collagen rSTH-Col.

[0041] Figure 4 SEM image of highly stable triple-helix recombinant collagen rSTH-Col;

[0042] Figure 5 Results of the high-stability triple-helix recombinant collagen rSTH-Col promoting HFF-1 cell proliferation;

[0043] Figure 6 Figure (a) and a bar chart (b) show the effect of highly stable triple-helix recombinant collagen rSTH-Col on the migration of HFF-1 cells. Detailed Implementation

[0044] To enable those skilled in the art to clearly and completely understand the technical solution of the present invention, the present invention will be further described in detail below with reference to embodiments. Obviously, the embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0045] This invention provides a recombinant collagen with a highly stable triple helix structure, the amino acid sequence of which contains a polypeptide chain composed of Gly-XY repeating units. By regulating the amino acid composition at the X and Y positions, the protein can still form a highly stable triple helix structure even without hydroxyproline.

[0046] Example 1: Preparation of highly stable triple-helix recombinant collagen rSTH-Col

[0047] (1) Gene construction

[0048] Design a nucleotide sequence that encodes the target recombinant collagen, with an amino acid sequence containing (Gly-XY). n The amino acid sequence of the highly stable triple-helix recombinant collagen rSTH-Col provided in this embodiment is shown in SEQ ID NO. 1. After codon preference optimization for E. coli, its nucleotide sequence is shown in SEQ ID No. 2.

[0049] (2) Construction of recombinant engineered bacteria

[0050] The nucleotide sequence of recombinant collagen rSTH-Col was synthesized and constructed into the E. coli expression vector pCold. The successful synthesis of the recombinant vector was confirmed by DNA sequencing. The recombinant vector was transformed into E. coli BL21-DE3 strain, and E. coli strains that highly expressed recombinant collagen rSTH-Col were screened. The successfully constructed strains were stored in glycerol at -80 °C.

[0051] (3) Protein expression

[0052] The engineered bacteria were inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37 °C until the logarithmic growth phase (OD600 was approximately 0.6–1.0). IPTG was added to a final concentration of 1 mM to induce expression, and the bacteria were cultured at 20–25 °C for 12–20 h.

[0053] (4) Protein purification

[0054] The bacterial cells were collected and resuspended in buffer solution, then subjected to high-pressure disruption and centrifugation to obtain the supernatant. The target recombinant collagen was purified by nickel affinity chromatography.

[0055] (5) Structure formation and post-processing

[0056] The purified protein was placed in a suitable buffer system and subjected to folding and self-assembly at low temperature to obtain recombinant collagen with a triple helix structure; the final product was then obtained by dialysis and freeze-drying.

[0057] Example 2: Characterization of recombinant collagen rSTH-Col

[0058] 1. SDS-PAGE characterization of recombinant collagen rSTH-Col

[0059] The purified recombinant collagen rSTH-Col was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), such as... Figure 1 As shown, the three batches of purified rSTH-Col appeared as a single band on the gel, with an apparent molecular weight of approximately 20 kDa, indicating that the present invention successfully prepared high-purity recombinant collagen rSTH-Col.

[0060] 2. Western Blot Detection of Recombinant Collagen rSTH-Col

[0061] The supernatant samples from the three rSTH-Col monoclonal antibodies (1, 2, and 3) after expression were lysed, mixed with loading buffer, and boiled for denaturation. After separation by SDS-PAGE electrophoresis, the samples were wet-transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h, followed by incubation overnight at 4 °C with anti-His tag primary antibody (1:3000). After washing with TBST, the membrane was incubated with HRP-labeled secondary antibody (1:5000) at room temperature for 2 h, and washed again. Finally, development and detection were performed using ECL chemiluminescent substrate. Results are as follows: Figure 2 As shown, a single clear band appears at the molecular weight (20kDa), indicating that the recombinant collagen rSTH-Col is successfully expressed, while the blank control group (WT) does not have this band.

[0062] 3. Circular dichroism characterization of recombinant collagen rSTH-Col

[0063] Circular dichroism spectroscopy is a commonly used method for characterizing collagen structure. Lyophilized recombinant collagen rSTH-Col was prepared as a 0.5 mg / mL aqueous solution. Using a cuvette with a 1 mm slit, circular dichroism spectroscopy was performed at 4 °C, scanning the entire wavelength range (190-260 nm) with a 1 nm wavelength interval and a dwell time of 5 s at each wavelength. The results are as follows: Figure 3 As shown in a, the recombinant collagen rSTH-Col prepared in this invention has a positive peak at 225 nm and a negative peak at 192 nm, which conforms to the CD characteristics of a typical triple helix structure of collagen, indicating that the recombinant collagen rSTH-Col has a triple helix structure.

[0064] The thermal degeneration temperature of the recombinant collagen rSTH-Col was measured under the following conditions: 4-20 °C, 1 °C / min; 20-50 °C, 0.3 °C / min; 50-80 °C, 1 °C / min. The results are as follows: Figure 3 As shown in b and 3c, the thermal distortion temperature of the recombinant collagen rSTH-Col prepared in this invention is 43.1 ℃.

[0065] 4. SEM characterization of recombinant collagen rSTH-Col

[0066] After incubation in PBS at pH 7.4 and 37 ℃ for a certain period, the assembled rSTH-Col protein was lyophilized. The lyophilized recombinant collagen rSTH-Col sample was then fixed to the scanning electron microscope stage using conductive adhesive. After sputtering a gold-palladium alloy conductive layer, the sample was observed under a scanning electron microscope. The results are as follows: Figure 4 As shown, under high magnification, the recombinant collagen rSTH-Col exhibits an interwoven fibrous network structure with continuous fibrous morphology and relatively uniform distribution. These results demonstrate that the recombinant collagen provided by this invention can form a higher-order structure with a fibrous morphology.

[0067] Example 3: Bioactivity evaluation of recombinant collagen rSTH-Col

[0068] 1. Cell proliferation experiment

[0069] 100 μL of a density of 1×10 5 HFF-1 cells per mL were added to 96-well plates and cultured at 37 °C in a 5% CO2 incubator for 24 h. The culture medium in the 96-well plates was then aspirated. Lyophilized recombinant type III collagen rSTH-Col was prepared into a protein solution with a concentration of 0.1 mg / mL using DMEM high-glucose medium, filtered through a 0.22 μm sterile filter for sterilization, and added to 96-well plates with replicates. The control group received only DMEM high-glucose medium. Cells were cultured at 37 °C in a 5% CO2 incubator for 24 h, 72 h, and 120 h, respectively. The proliferative effect of recombinant collagen rSTH-Col on human skin fibroblasts (HFF-1) was detected using a CCK-8 assay.

[0070] like Figure 5 As shown, the recombinant collagen rSTH-Col has a significant effect on promoting the proliferation of human skin fibroblasts HFF-1. After 3 and 5 days of culture, the relative growth rate of cells with recombinant collagen rSTH-Col is significantly better than that of the control group.

[0071] 2. Cell migration experiment

[0072] The cell migration-promoting effect of recombinant collagen rSTH-Col was determined using the cell scratch assay. 2 mL of rSTH-Col with a density of 2.5 × 10⁻⁶ was used. 5HFF-1 cells were added at a density of 10 cells / mL to 6-well plates and cultured for 24 h until cell confluence reached 95%-100%. Using a pipette tip with a 200 μL range, a longitudinal scratch was made on the bottom of the 6-well plate using a ruler. The culture medium was then aspirated. Lyophilized recombinant collagen rSTH-Col, tissue-extracted bovine type III collagen Col III, and recombinant expressed single-chain type III collagen rColIII were each prepared into a 0.1 mg / mL protein solution using DMEM high-glucose medium, and 2 mL of each solution was added to the corresponding well of the 6-well plate. 2 mL of DMEM high-glucose medium was added to one well of the 6-well plate as a control. The changes in scratch area were recorded under an inverted microscope at 0 h and 24 h.

[0073] like Figure 6 As shown, compared with the blank control group, the recombinant collagen rSTH-Col exhibited a good cell migration-promoting effect.

[0074] In summary, this invention is based on the Gly-XY repeat sequence and obtains recombinant collagen rSTH-Col through gene recombination expression. This recombinant collagen can form a stable triple helix conformation without hydroxyproline modification, and its thermal denaturation temperature can reach 43.1 °C. Simultaneously, the protein exhibits good water solubility and structural homogeneity, and can self-assemble into a fibrous morphology in vitro. Therefore, this recombinant collagen can be used as a biomaterial in tissue engineering scaffolds, skin repair materials, and related medical materials. Compared with animal-derived collagen, the recombinant collagen of this invention has the characteristics of well-defined composition and good batch-to-batch consistency, making it suitable for large-scale preparation.

[0075] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A highly stable triple-helix recombinant collagen, characterized in that, The recombinant collagen comprises a polypeptide chain consisting of Gly-XY repeating sequences, wherein: ① The amino acid at position X is selected from one or more of Pro, Asp, and Glu; ② The amino acid at the Y position is selected from one or more of Pro, Lys, and Arg; Furthermore, the recombinant collagen satisfies the following characteristics: (1) Contains no hydroxyproline; (2) It can form a triple helix conformation in aqueous solution; (3) Its thermal denaturation temperature Tm ≥ 40 ℃; (4) It can self-assemble into a fibrous structure under physiological conditions.

2. The recombinant collagen according to claim 1, characterized in that, The Gly-XY repeat sequence is (Gly-XY). n , where n is 10~30.

3. The recombinant collagen according to claim 2, characterized in that, The sequence contains (Gly-Pro-Lys-Gly-Asp-Pro). n Structural unit.

4. The recombinant collagen according to claim 1, characterized in that, The amino acid sequence of the recombinant collagen is as shown in SEQ ID NO.1 or a variant thereof with the same function.

5. A method for constructing the recombinant collagen according to any one of claims 1 to 4, characterized in that, By regulating the composition and arrangement of amino acids at the X and Y positions in the Gly-XY repeat sequence, the recombinant collagen can form a stable triple helix structure under conditions without hydroxyproline.

6. A method for preparing the recombinant collagen according to any one of claims 1 to 4, characterized in that, include: (1) Construct the nucleotide sequence encoding the recombinant collagen; (2) The nucleotide sequence is ligated into an expression vector and transformed into a host cell; (3) The recombinant collagen was obtained by inducing expression and purification.

7. The preparation method according to claim 6, characterized in that, The expression vector includes pCold or pET, and / or the host cell includes Escherichia coli or yeast.

8. A recombinant vector or recombinant genetically engineered bacterium comprising a nucleotide sequence encoding the recombinant collagen of any one of claims 1 to 4.

9. The use of the recombinant collagen according to any one of claims 1 to 4 in the preparation of biomaterials.

10. The application according to claim 9, characterized in that, The biomaterials include tissue engineering scaffolds, skin repair materials, or medical biomaterials.

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