CN116355868A - High-expression high-activity proline hydroxylase L593m, coding gene and application thereof in catalyzing hydroxylation of collagen - Google Patents
High-expression high-activity proline hydroxylase L593m, coding gene and application thereof in catalyzing hydroxylation of collagen Download PDFInfo
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Abstract
The invention discloses a proline hydroxylase L593m with high expression and high activity, which is characterized in that the amino acid sequence is shown as SEQ ID No. 3. Also discloses the coding gene, the expression vector and the expression host bacterium and the application thereof in catalyzing the hydroxylation of collagen. The invention provides a proline 4-hydroxylase variant L593m with high expression and activity, the amino acid sequence of which is shown as SEQ NO.3, and the nucleotide sequence of which is shown as SEQ 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
The invention relates to a proline hydroxylase L593m with high expression and high activity, a coding gene and application thereof in catalyzing hydroxylation of collagen, and belongs to the technical field of protein expression.
Background
Collagen is a biological macromolecular protein, is a main protein component of connective tissue in an animal body, is a functional macromolecular protein with the greatest content and the greatest distribution in the mammal body, accounts for 30% -40% of the total protein of the body, and even reaches more than 80% in some organisms. Collagen has strong biological activity and biological function, participates in migration, differentiation and proliferation of cells in skin, tendons and other connective tissues of human body, is a promoter of metabolism, and can slow down aging and hypofunction of biological tissues. Thus, collagen is particularly important for improving the quality of life health in the elderly, in climacteric women, in the case of hyperkinesia, in the case of fractures or in the case of joint pain. With age, collagen is gradually lost (1% of collagen is lost every year), so that intercellular mucopolysaccharide is reduced, connective tissue elasticity is weakened, and skin loses elasticity, becomes thinner and ages. Collagen reduction also leads to dermis fiber rupture, lipoatrophy, sweat gland and sebaceous gland secretion reduction, and finally skin dryness, wrinkle increase, color spots and other 'aging' manifestations. This aging phenomenon is continuously aggravated with age, and visually manifests as wrinkles and age spots on the face, darkening and loss of gloss of the skin, rough skin and poor elasticity, arteriosclerosis, senile cataract of the eye lens, reduced corneal transparency, inflexibility of the arms and legs, and even joint pain. Therefore, the collagen has wide application in the fields of medical treatment, skin care, health care products and the like. The efficient synthesis of collagen has important market value.
Collagen needs to be improved in stability and crosslinking effect by hydroxylation of proline and lysine during synthesis. Hydroxylation of proline residues of collagen is critical for stabilization of the triple helix structure of collagen. Mammalian prolyl 4-hydroxylase (P4H) is an enzyme with an α2β2 tetrameric arrangement, a key enzyme for collagen synthesis, contributing to post-translational hydroxylation of proline residues in the collagen chain. Since human proline 4-hydroxylase requires two protein subunits to make up the tetramer, this makes in vitro and in vivo proline hydroxylation of collagen very difficult, which severely hampers the progress of collagen synthesis. In recent years, there have also been team attempts to use the proline hydroxylase L593 (Shi, J., ma, X., gao, Y.et. Hydroxylation of Human Type III Collagen Alpha Chain by Recombinant Coexpression with aViral Prolyl 4-Hydroxylase in Escherichia coll.protein J36, 322-331 (2017): https:// doi.org/10.1007/s 10930-017-9723-0) of a minivirus to effect hydroxylation in collagen. The method can realize the effect of human proline 4-hydroxylase tetramer only by expressing or purifying the L593 monomer, thereby greatly reducing the difficulty of hydroxylation of collagen. However, expression of L593 in cells is extremely difficult and very detrimental to collagen synthesis.
Disclosure of Invention
The invention discloses a high-expression high-activity proline hydroxylase L593m, the amino acid sequence of which is shown as SEQ ID No. 3; or has more than 95% homology with SEQ ID No.3, and has substantially the same enzyme activity as the proline hydroxylase L593m shown in SEQ ID No. 3.
The invention also discloses a coding gene of the proline hydroxylase L593 m.
Preferably, the nucleotide sequence is shown as SEQ ID No.4.
The invention also discloses an expression vector of the proline hydroxylase L593 m.
Preferably, the vector is pGEX-6P1.
The invention also discloses an expression host bacterium of the proline hydroxylase L593 m.
And the application of the proline hydroxylase L593m in catalyzing collagen hydroxylation.
The invention also discloses an expression method of the collagen, which is characterized by comprising the following steps:
(1) Constructing a collagen expression vector, and transfecting the collagen expression vector and the expression vector into host bacteria together;
(2) Inducing expression of collagen and proline hydroxylase L593 m;
(3) Collagen is separated and purified by using a protein purification technology.
Preferably, the amino acid sequence of the collagen is shown as SEQ ID No. 5.
Preferably, the host bacterium is E.coli.
The invention provides a proline 4-hydroxylase variant L593m with high expression and activity, the amino acid sequence of which is shown as SEQ ID NO.3, and the nucleotide sequence of which 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.
Drawings
FIG. 1 is a schematic protein structure of proline hydroxylase L593.
FIG. 2 is a schematic representation of the protein structure of proline hydroxylase L593 m.
FIG. 3 is a SDS-PAGE map of induced expression of L593 and L593 m.
FIG. 4 shows the separation and purification of collagen after co-expression of L593 and L593m with collagen.
FIG. 5 is a graph showing the measurement of collagen expression and proline hydroxylation.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.
Unless defined otherwise, 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 belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention.
The materials or instruments used in the following examples, if not specifically described, were available from conventional commercial sources.
Example 1
Structural prediction was performed on L593, and the prediction results of AlphaFold2 used by the prediction tool are shown in fig. 1. According to the result of protein structure prediction, the L593 (the amino acid sequence is shown as SEQ ID NO:1, the DNA sequence is shown as SEQ ID NO: 2) is subjected to enzyme activity and stability modification, and the modified L593m (the amino acid sequence is shown as SEQ ID NO:3, the DNA sequence is shown as SEQ ID NO: 4) protein structure prediction is shown as figure 2.
Example 2
In order to verify the activity of the transformed L593, sequences of SEQ ID NO. 2 and SEQ ID NO.4 were obtained by gene synthesis and respectively constructed into pGEX-6P1 expression vectors. The constructed vector was transformed into Rosetta (DE 3) E.coli competence. Culturing in liquid LB culture medium containing corresponding resistance to OD600 value of 0.4-1.0. The recombinant strain containing the expression vector was induced to express using 1mM IPTG for 4 hours. Induced expression results were detected using SDS-PAGE. As a result, as can be seen from FIG. 3, the expression level of L593m was significantly higher than that of unmodified L593 as can be seen from FIG. 3.
Example 3
To further verify the hydroxylation activity of L593m on collagen proline. We constructed expression vectors for human type III collagen. The amino acid sequence of the collagen is shown as SEQ ID NO.5, and the DNA sequence is shown as SEQ ID NO. 6. We transformed the collagen expression vector and the L593m expression vector into Rosetta (DE 3) e.coli competence. Culturing in liquid LB culture medium containing corresponding resistance to OD600 value of 0.4-1.0. The recombinant strain containing the expression vector was induced to express using 1mM IPTG and cultured at 37℃at 200rpm for 4-6 hours. And (5) centrifuging at 10000rpm at 4 ℃ for 20min, and collecting the thalli. The cells were washed 2 times with PBS. The cells were resuspended in lysis buffer (20mM Tris,350mM NaCl,pH 8.0), disrupted using pressure or ultrasound, stained with a microscope until no apparent cells were present. Centrifugation was performed at 12000rpm at 4℃for 20min, and the supernatant was collected and filtered through a 0.45um filter. After the Ni-NTA affinity chromatographic column is fully balanced by using 20 times of column volume of lysis buffer, the filtered bacterial lysate is added, the flow rate is 0.5ml/min, and then the column is fully cleaned by using 10 times of column volume of lysis buffer containing 40mM imidazole. The target protein was eluted by adding three column volumes containing 40mM imidazole lysis buffer. The cut flow-through was collected, centrifuged at 10000rpm with a 3kDa ultrafiltration tube at 4℃for 10min, and 5 volumes of the diluted solution (50mM Tris,100mM NaCl,pH 7.5) were added. SDS-PAGE showed the result of protein purification, and the result is shown in FIG. 4. We used the proline hydroxylation assay kit (Soxhibao, cat. BC 0250) to measure the degree of hydroxylation of proline in purified collagen, the results of which are shown in FIG. 5. The modified L593m has better proline hydroxylation activity.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A high-expression high-activity proline hydroxylase L593m is characterized in that the amino acid sequence is shown in SEQ ID No. 3.
2. The proline hydroxylase L593m encoding gene of claim 1.
3. The coding gene according to claim 2, wherein the nucleotide sequence is shown in SEQ ID No.4.
4. The expression vector of proline hydroxylase L593m of claim 1.
5. The expression vector of claim 4, wherein the vector is pGEX-6P1.
6. The expression host bacterium of proline hydroxylase L593m of claim 1.
7. Use of the proline hydroxylase L593m of claim 1 for catalyzing hydroxylation of collagen.
8. A method for expressing collagen, comprising the steps of:
(1) Constructing a collagen expression vector, and transfecting the collagen expression vector and the expression vector into host bacteria;
(2) Inducing expression of collagen and proline hydroxylase L593 m;
(3) Collagen is separated and purified by using a protein purification technology.
9. The method for expressing collagen according to claim 8, wherein 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, wherein the host bacterium is escherichia coli.
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| CN117624342A (en) * | 2023-10-31 | 2024-03-01 | 广州市金因源生物技术有限公司 | Preparation method of hydrolyzed collagen and application of hydrolyzed collagen on skin |
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