High expression of high activity proline hydroxylase l593m, encoding gene and its application in catalyzing collagen hydroxylation

Abstract

The application discloses a high-expression and high-activity proline hydroxylase L593m, characterized in that the amino acid sequence is shown as SEQ ID No. 3. The application also discloses an encoding gene, an expression vector, an expression host bacterium and application in catalyzing collagen hydroxylation. The application provides a high-efficiency expression and activity proline 4-hydroxylase variant L593m, the amino acid sequence of which is shown as SEQ ID No. 3, and the nucleotide sequence is shown as SEQ ID No. 4. Compared with L593, the proline 4-hydroxylase variant L593m has the characteristics of simple expression, better activity, easier purification, higher yield and the like, can be used for in-vivo hydroxylation of collagen, and is suitable for industrial production and application.

Description

Technical Field

[0001] This invention relates to a highly expressed and highly active proline hydroxylase L593m, its encoding gene, and its application in catalyzing collagen hydroxylation, belonging to the field of protein expression technology. Background Technology

[0002] Collagen is a large biological protein molecule, a major protein component of connective tissue in animals, and the most abundant and widely distributed functional protein in mammals, accounting for 30%–40% of total body protein, and even exceeding 80% in some organisms. Collagen possesses strong biological activity and function, participating in cell migration, differentiation, and proliferation in human skin, tendons, and other connective tissues. It acts as a metabolic promoter, slowing down tissue aging and functional decline. Therefore, collagen is particularly important for improving the quality of life and health of the elderly, menopausal women, those who overexert themselves, those with fractures, or those prone to joint pain. With age, collagen is gradually lost (approximately 1% of collagen is lost annually), leading to a reduction in intercellular mucopolysaccharides, decreased elasticity of connective tissue, and loss of skin elasticity, thinning, and aging. Reduced collagen also causes dermal fiber breakage, fat atrophy, and decreased secretion from sweat and sebaceous glands, ultimately resulting in signs of aging such as dry skin, increased wrinkles, and age spots. This aging process intensifies with age, manifesting as wrinkles and age spots on the face, dull and lackluster skin, rough skin with reduced elasticity, arteriosclerosis, age-related cataracts in the eye, decreased corneal transparency, and decreased flexibility in the arms and legs, even joint pain. Therefore, collagen has wide applications in the medical, skincare, and health product fields. The efficient synthesis of collagen has significant market value.

[0003] During collagen synthesis, hydroxylation of proline and lysine residues is necessary to enhance its stability and cross-linking properties. Hydroxylation of proline residues in collagen is crucial for the stability of its triple helix structure. Mammalian prolyl 4-hydroxylase (P4H), an enzyme with an α2β2 tetrameric arrangement, is a key enzyme in collagen synthesis, playing a role in the post-translational hydroxylation of proline residues in the collagen chain. Because human prolyl 4-hydroxylase requires two protein subunits to form a tetramer, the in vitro and in vivo proline hydroxylation of collagen is extremely difficult, severely hindering the progress of collagen synthesis. In recent years, some teams have attempted to use the proline hydroxylase L593 from a small virus (Shi, J., Ma, X., Gao, Y. et al. Hydroxylation of Human Type III Collagen Alpha Chain by Recombinant Coexpression with aViral Prolyl 4-Hydroxylase in Escherichiacoli. Protein J 36, 322–331 (2017). https: / / doi.org / 10.1007 / s10930-017-9723-0) to achieve in vivo hydroxylation of collagen. This method only requires the expression or purification of L593 monomers to achieve the effect of human proline 4-hydroxylase tetramer, thus greatly reducing the difficulty of collagen hydroxylation. However, the expression of L593 in cells is extremely difficult, which is very unfavorable for collagen synthesis. Summary of the Invention

[0004] This invention discloses a highly expressed and highly active proline hydroxylase L593m, the amino acid sequence of which is shown in SEQ ID No. 3; or has more than 95% homology with SEQ ID No. 3 and has essentially the same enzyme activity as the proline hydroxylase L593m shown in SEQ ID No. 3.

[0005] The present invention also discloses the encoding gene of the above-mentioned proline hydroxylase L593m.

[0006] Preferably, its nucleotide sequence is shown in SEQ ID No. 4.

[0007] The present invention also discloses the expression vector of the above-mentioned proline hydroxylase L593m.

[0008] Preferably, the carrier is pGEX-6P1.

[0009] The present invention also discloses the expression host bacteria of the above-mentioned proline hydroxylase L593m.

[0010] And the application of the aforementioned proline hydroxylase L593m in catalyzing collagen hydroxylation.

[0011] This invention also discloses a method for expressing collagen, characterized by the following steps:

[0012] (1) Construct a collagen expression vector and co-transfect it into the host bacteria along with the above expression vector;

[0013] (2) Inducing the expression of collagen and proline hydroxylase L593m;

[0014] (3) Collagen was separated and purified using protein purification technology.

[0015] Preferably, the amino acid sequence of the collagen is shown in SEQ ID No. 5.

[0016] Preferably, the host bacterium is Escherichia coli.

[0017] This invention provides a highly efficient proline 4-hydroxylase variant L593m, the amino acid sequence of which is shown in SEQ ID NO.3 and the nucleotide sequence in SEQ ID NO.4. Compared to L593, the proline 4-hydroxylase variant L593m has the advantages of simple expression, better activity, easier purification, and higher yield, and can be used for in vivo hydroxylation of collagen, making it suitable for industrial production and application. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the protein structure of proline hydroxylase L593.

[0019] Figure 2 This is a schematic diagram of the protein structure of proline hydroxylase L593m.

[0020] Figure 3 SDS-PAGE images of induced expression of L593 and L593m.

[0021] Figure 4 Collagen was isolated and purified after L593 and L593m were co-expressed with collagen.

[0022] Figure 5 This is a graph showing the expression of collagen and the degree of proline hydroxylation. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments, but the description of the embodiments does not limit the scope of protection of the present invention in any way.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0025] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.

[0026] Example 1

[0027] Structural predictions were performed on L593 using AlphaFold2. The prediction results are shown below. Figure 1 Based on the protein structure prediction results, the enzyme activity and stability of L593 (amino acid sequence shown in SEQ ID NO:1, DNA sequence shown in SEQ ID NO:2) were modified. The predicted protein structure of the modified L593m (amino acid sequence shown in SEQ ID NO:3, DNA sequence shown in SEQ ID NO:4) is shown below. Figure 2 .

[0028] Example 2

[0029] To verify the activity of the modified L593, the sequences of SEQ ID NO:2 and SEQ ID NO:4 were obtained through gene synthesis and constructed into the pGEX-6P1 expression vector. The constructed vectors were transformed into competent Rosetta(DE3) Escherichia coli cells. The cells were cultured in liquid LB medium containing the corresponding antibiotics until the OD600 value reached 0.4–1.0. The recombinant strains containing the expression vectors were induced to express the gene using 1 mM IPTG for 4 h. The induction expression results were detected using SDS-PAGE. Results are shown below. Figure 3 ,from Figure 3 It can be seen that the expression level of L593m is significantly higher than that of the unmodified L593.

[0030] Example 3

[0031] To further verify the hydroxylation activity of L593m on proline in collagen, we constructed an expression vector for human type III collagen. The amino acid sequence of collagen is shown in SEQ ID NO:5, and its DNA sequence is shown in SEQ ID NO:6. We transformed the collagen expression vector and the L593m expression vector into competent cells of *E. coli* (DE3). The cells were cultured in liquid LB medium containing the appropriate antibiotics until the OD600 reached 0.4–1.0. The recombinant strain containing the expression vector was induced to express the cells with 1 mM IPTG and cultured at 37°C and 200 rpm for 4–6 h. The cells were then collected by centrifugation at 10,000 rpm and 4°C for 20 min. The cells were washed twice with PBS. The cells were resuspended in lysis buffer (20 mM Tris, 350 mM NaCl, pH 8.0) and disrupted by pressure or sonication. Microscopic staining was performed until no obvious cells were observed. Centrifuge at 12000 rpm, 4°C for 20 min, collect the supernatant, and filter through a 0.45 μm filter membrane. Equilibrate the Ni-NTA affinity chromatography column with 20 column volumes of lysis buffer, add the filtered bacterial lysis buffer, and wash thoroughly with 10 column volumes of lysis buffer containing 40 mM imidazole at a flow rate of 0.5 ml / min. Elute the target protein with 3 column volumes of lysis buffer containing 40 mM imidazole. Collect the flow-through after cleavage, centrifuge through a 3 kDa ultrafiltration tube at 10000 rpm, 4°C for 10 min, and add 5 volumes of dilution buffer (50 mM Tris, 100 mM NaCl, pH 7.5). Analyze the protein purification results using SDS-PAGE; see the attached table. Figure 4 We used a proline hydroxylation assay kit (Solepro, catalog number BC0250) to determine the degree of proline hydroxylation in purified collagen. The results are shown in the table below. Figure 5 The modified L593m exhibits better proline hydroxylation activity.

[0032] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A highly expressed and highly active proline hydroxylase L593m, characterized in that... Its amino acid sequence is shown in SEQ ID No.

3.

2. The gene encoding the proline hydroxylase L593m as described in claim 1.

3. The encoding gene according to claim 2, characterized in that... Its nucleotide sequence is shown in SEQ ID No.

4.

4. The expression vector for proline hydroxylase L593m as described in claim 1.

5. The expression vector according to claim 4, characterized in that... The carrier is pGEX-6P1.

6. The expression host bacteria of proline hydroxylase L593m as described in claim 1.

7. The application of the proline hydroxylase L593m according to claim 1 in catalyzing the hydroxylation of collagen.

8. A method for expressing collagen, characterized in that... The steps include: (1) Construct a collagen expression vector and co-transfect it into the host bacteria along with the expression vector described in claim 4; (2) Inducing the expression of collagen and proline hydroxylase L593m; (3) Collagen was separated and purified using protein purification technology.

9. The method for expressing collagen according to claim 8, characterized in that... The amino acid sequence of the collagen is shown in SEQ ID No.

5.

10. The method for expressing collagen according to claim 8 or 9, characterized in that... The host bacterium is Escherichia coli.

Images