BaP4H mutant and application thereof

Abstract

The invention provides a BaP4H mutant and application thereof, and belongs to the technical field of enzyme engineering, the BaP4H mutant is mutated on an amino acid sequence as shown in SEQ ID NO.1; the invention further provides a recombinant expression vector for expressing the BaP4H mutant, a recombinant cell, a preparation method and application. According to the BaP4H mutant provided by the invention, the recombinant collagen proline is efficiently hydroxylated, and the stability of the recombinant collagen is enhanced.

Description

[0001] This application is a divisional application with a filing date of July 31, 2024, application number 202411037202.3, and invention name “A BaP4H mutant, preparation method and application thereof”. Technical Field

[0002] The invention belongs to the technical field of enzyme engineering, and in particular relates to a BaP4H mutant and application thereof. Background Art

[0003] Proline hydroxylation is the most common post-translational modification in collagen. The resulting product, trans-4-hydroxyproline (Hyp), is essential for the stability and function of collagen. Studies have shown that the lack of proline hydroxylation in collagen can destabilize the triple helix structure and affect its binding to integrins. 4-Proline hydroxylases (P4Hs) are a class of oxidases that depend on ferrous ions and 2-oxoglutarate and can catalyze the formation of Hyp. P4H (BaP4H) from Bacillus anthracis can modify collagen-like proline-rich peptides.

[0004] The triple helix structure of collagen contains a rich and highly repetitive tripeptide motif: Gly-XY, where the X and Y positions are usually proline and hydroxyproline. In vivo, the proline at the X position needs to be hydroxylated to generate 4-hydroxyproline to form a stable collagen triple helix structure.

[0005] At present, some progress has been made in the co-expression of collagen genes and P4Hs genes in yeast to achieve the production of hydroxylated collagen in an industrial expression system. However, wild-type P4Hs has problems such as low expression level, low hydroxylation efficiency of collagen, and no improvement in the stability of recombinant collagen after hydroxylation. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a BaP4H mutant. Experimental results show that the mutant can efficiently hydroxylate recombinant collagen and enhance the stability of the recombinant collagen.

[0007] The first object of the present invention is to provide a BaP4H mutant, which is a mutation of the amino acid sequence shown in SEQ ID NO.1, including deletion and substitution.

[0008] Preferably, the modified position and the amino acids before and after the mutation are selected from any one of the following:

[0009] The group consisting of: N3S, N4S, N5S, I7M, G8N, E9R, N10E, K11I, E12K, T14E, I15R, D17A, H18D, K19D, G20E, N21S, I23H, K24T, T25A, E26R, D27 deletion, R28K, E29A, I30F, I32 deletion, I33 deletion, S34E, K35G, E37S, E38N, L40M, I41P, L44Y, G45Y, N46A, L48S, S49E, D50A, E51W, E52 deletion, D54A, E55V, L56N, I57K, E58W, L59Q , S60A, K61 deletion, S62E, K63L, L64T, A65N, R66P, S67A, K68A, G70T, S71L, S72 deletion, R73T, D74A, N76S, D77P, I78A, R79A, R79T, R79S, R79L, S81L, S82 deletion, G83N, A84T, L86K, D87K, D88V, N89E, E90S, L91V, T92M, A93D, K94L, I95V, E96M, K97Q, R98S, I99D, S100 deletion, S101A, I102A, N104Q, A107T, S108K , H109L, G110S, E111M, G112A, L113A, H114D, I115E, L116 deletion, N117S, E119K, D121G, K125Y, A126W, H127L, D129K, A132Q, E133V, H134T, H134D, H134S, H134K, S135N, R136E, S137M, A138N, A139R, N140S, N141E, R142E, I143S, S144V, T145D, L146K, L150Y, N151Q, D152G, E154A, E155S, G156N , G157M, E158A, T159D, K163S, L164V, N165W, L166Q, S167H, H169I, R171I, K172A, G173R, A175T, E179P, D184W, S186F, L187K, N188A, E189V, L190I, T191P, L192G, H193A, G194M, G195 deletion, A196C, T199S, K200V, G201M, E202Q, I205V, A206K, T207S, R211A, R212Y, G213P, T214Q, and E217N, and combinations thereof.

[0010] More preferably, the mutant wherein each modification is independently a substitution or a deletion, wherein the variant has at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the mature polypeptide of SEQ ID NO.1.

[0011] The second object of the present invention is to provide an expression system of the BaP4H mutant, including but not limited to nucleic acid molecules, recombinant expression vectors, and recombinant cells.

[0012] Preferably, the nucleic acid molecule comprises a nucleotide sequence encoding the BaP4H mutant or its complementary sequence.

[0013] Preferably, the recombinant expression vector contains the nucleic acid molecule. The expression vector includes any nucleic acid molecule (e.g., plasmid, cosmid, virus, self-replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule) derived from any source and capable of genome integration or autonomous replication, which includes nucleic acid molecules of one or more nucleic acid molecules that are operably connected. The carrier may include, for example, one or more selectable markers, one or more replication origins (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or promotes the stable integration of constructs into the elements in the host cell genome.

[0014] Preferably, the recombinant cell contains the recombinant expression vector, or the exogenous nucleic acid molecule is integrated into its chromosome.

[0015] The third object of the present invention is to provide an enzyme preparation comprising the BaP4H mutant.

[0016] The fourth object of the present invention is to provide the use of the aforementioned BaP4H mutant, nucleic acid molecule, recombinant expression vector, recombinant cell, and enzyme preparation containing the BaP4H mutant in catalyzing the proline hydroxylation of recombinant collagen.

[0017] The specific technical solutions of the present invention are as follows:

[0018] The present invention provides a BaP4H mutant, which at least comprises a mutation site in which aspartic acid at position 50 of the polypeptide shown in SEQ ID NO.1 is mutated to alanine.

[0019] Preferably, the mutation site is: D50A+R73T, D50A+D129K, D50A+D152G, D50A+H134T, D50A+H114D, D50A+R142E or D50A+S81L.

[0020] Preferably, when the mutation site is D50A+R73T, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.3; or, when the mutation site is D50A+D129K, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.5; or, when the mutation site is D50A+D152G, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.7; or, when the mutation site is D50A+H134T, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.9; or, when the mutation site is D50A+H114D, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.11; or, when the mutation site is D50A+R142E, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.13; or, when the mutation site is D50A+S81L, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID Shown in NO.15.

[0021] Preferably, the present invention provides a gene encoding the above-mentioned BaP4H mutant, specifically: the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.3 is shown in SEQ ID NO.2; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.5 is shown in SEQ ID NO.4; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.7 is shown in SEQ ID NO.6; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.9 is shown in SEQ ID NO.8; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.11 is shown in SEQ ID NO.10; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.13 is shown in SEQ ID NO.12; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.15 is shown in SEQ ID NO.14.

[0022] Preferably, the present invention provides a recombinant expression vector containing the above encoding gene, wherein the vector is pGRO7, and a strep-tag is added to pGRO 7.

[0023] Preferably, the present invention provides a recombinant cell, wherein the recombinant cell contains the above-mentioned recombinant expression vector, or the above-mentioned exogenous encoding gene is integrated into its chromosome.

[0024] Preferably, the present invention provides an enzyme preparation, which comprises the above-mentioned BaP4H mutant.

[0025] Preferably, the present invention also provides the use of the above-mentioned BaP4H mutant, or the above-mentioned encoding gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned enzyme preparation in catalyzing the proline hydroxylation of recombinant collagen.

[0026] Preferably, the application is to co-express the BaP4H mutant with recombinant collagen to catalyze the hydroxylation of proline in the recombinant collagen, comprising the following steps:

[0027] i) constructing the coding gene of the BaP4H mutant into the pGRO7-strep plasmid and the coding gene of the recombinant collagen into the pET28a plasmid, mixing them and co-transforming them into Escherichia coli Rosetta competent cells, applying them on double-antibody LB solid culture medium and culturing them overnight;

[0028] ii) picking up a single colony grown on the plate and culturing it in liquid SOC dual-antibody culture medium as a seed solution;

[0029] iii) The seed solution was inoculated into the double-resistance LB liquid culture medium, and then cultured for 1 hour, and arabinose with a final concentration of 2 mg / mL was added, and IPTG with a final concentration of 1 mM was added to induce the culture.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides a BaP4H mutant, which can efficiently hydroxylate recombinant collagen and further enhance the stability of recombinant collagen; the present invention also optimizes the conditions for culturing recombinant cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It shows that different co-expression conditions of BaP4H and recombinant collagen affect the structure of recombinant collagen.

[0033] Figure 2 Shown are the SDS-PAGE verification results of recombinant collagen after co-expression of BaP4H mutant and recombinant collagen.

[0034] Figure 3 Shown are the Western Blot-strep validation results of the BaP4H mutant after co-expression of the BaP4H mutant with recombinant collagen.

[0035] Figure 4 Shown are the results of stability assay of recombinant collagen after hydroxylation with BaP4H mutants. DETAILED DESCRIPTION

[0036] The present invention does not specifically limit the preparation method of the recombinant vector, and the conventional preparation method of the recombinant vector in the art can be used. In the present invention, the gene can be synthesized by a biotechnology company. The present invention does not specifically limit the separation and purification method, and the conventional protein separation and purification method in the art can be used; the preferred technical scheme is described in the examples.

[0037] The present invention characterizes the structure of collagen by circular dichroism (CD) commonly used in the art. CD is a spectroscopic method used to determine the structure of compounds with chiral structures that can produce left-right optical differential absorption, and is mainly used to determine the asymmetry of molecular structures. Generally, biomacromolecules contain chiral groups and structures, so CD is often used to measure and observe changes in the structure and conformation of biomacromolecules. The CD characteristics of the triple helix structure of collagen generally have a positive absorption peak near 221nm and a negative absorption peak near 195nm (industry standard YY / T1849-2022). The position of the absorption peak will shift with the change of amino acid sequence and length. The thermal stability of proteins is generally expressed by the midpoint temperature of thermal denaturation (melting temperature, Tm), that is, the temperature at which the protein unfolds 50%. For collagen, it refers to the temperature at which the triple helix structure unwinds, each forming a single chain, and the triple helix unwinds to 50%. Therefore, the CD spectrum can be used to study the helical structure of collagen and its thermal denaturation process.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1. Optimization of co-expression conditions of BaP4H and recombinant collagen SEQ9

[0040] The pGRO-strep-BaP4H plasmid and pET28a-SEQ9 plasmid were constructed and electroporated into Rosetta competent cells and plated on solid LB plates (Cm + The wild-type BaP4H single colony grown on the plate was picked into 4 mL of liquid SOC medium (Chloramphenicol final concentration of 34 μg / mL) and cultured overnight at 37°C. The plasmid was extracted by culturing overnight at 37°C. The pET28a-SEQ9 plasmid was extracted in the same way. pGRO-strep-BaP4H and pET28a-SEQ9 were co-transfected into Rosetta competent cells and spread on double-antibody LB solid medium (Chloramphenicol final concentration of 34 μg / mL, Kanamic final concentration of 50 μg / mL) for overnight culture.

[0041] Pick the single colony grown on the plate into 10mL double-antibody liquid LB medium (Chloramphenicol final concentration 34μg / mL, Kanamic final concentration 50μg / mL), and culture at 37℃ overnight. Transfer 10mL of bacterial liquid to 100mL double-antibody liquid LB medium and culture at 37℃ overnight. Transfer 10mL of the liquid strain activated overnight to 1L liquid LB medium at a 1% inoculation rate, divide into 7 groups, and induce expression according to the culture conditions 1-7 in the table:

[0042]

[0043] 7000rmp, 4℃ centrifuge for 30min to collect bacteria. Add Lysis Buffer according to the amount of bacteria: Lysis Buffer = 1: 8 (W / V) to re-dissolve the bacteria, and use a high-pressure homogenizer to break the cells. Break the whole bacteria at 18000rpm, 45min, centrifuge at 4℃, filter the supernatant through a 0.45μm filter, add 1mL Ni-beads per 1L of bacterial solution, purify the protein by affinity chromatography, and dialyze with potassium phosphate buffer after purification. The dialyzed protein is measured by CD using a circular dichroism spectrometer.

[0044] The experimental results are as follows Figure 1 As shown, adding 2 mg / mL arabinose 1 hour after amplification had the best effect, and subsequent co-expression was performed under this condition.

[0045] Example 2: Co-expression of BaP4H mutant and recombinant collagen protein Recombinant collagen protein SEQ9

[0046] 1. The mutant constructed in the present invention is a mutant of wild-type BaP4H, and the amino acid sequence of the wild-type BaP4H is as follows:

[0047] MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSDEECDELIELSKSKLARSKVGSSRDVNDIRTSSGAFLDDNELTAKIEKRISSIMNVPASHGE GLHILNYEVDQQYKAHYDYFAEHSRSAANNRISTLVMYLNDVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYKEF(SEQ ID NO.1).

[0048] 2. The mutants constructed by the present invention and their numbers are shown in the following table:

[0049] Mutant number Mutation site B23 D17A C4 D17A+S67A C5 D17A+N21S C6 D17A+I102A C7 D17A+R211A C8 D50A+R73T C10 D50A+D129K C11 D50A+D152G C13 D50A+H134T C14 D50A+H114D C15 D50A+R142E C17 D50A+S81L

[0050] 3. The nucleotide sequence and amino acid sequence involved in the present invention are as follows:

[0051] (1) When the mutation site is D50A+R73T:

[0052] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO.2):

[0053] atgacaaacaacaatcaaataggtgaaaataaggaacaaactatttttgatcataaaggaaatgtaattaagacagaggatagagaaatac

[0054] aaattatttcaaaattcgaagaacctcttattgtcgtgttaggaaatgtattaagtgcagaagagtgtgatgaattaattgaattgtctaaaagt

[0055] aaattagcacgttcaaaagttggttcatcaactgatgtaaatgatattcgaacgagtagtggtgcatttttggacgataatgaacttacggcg

[0056] aagattgaaaaacggatttcatctatcatgaatgttcctgcgtcgcatggagaaggattacacattttaaattatgaagtggatcaacaatata

[0057] aagcgcattatgattattttgcggaacatagtagatccgctgctaataatcgtattagtacgcttgttatgtacttaaatgatgtcgaagaagg

[0058] cggagaaacgttctttccgaaattaaatctttctgtgcaccctagaaagggaatggcagtatactttgagtatttctatcaagaccaatcatta

[0059] aacgagcttacgttacacggaggggcacctgtaacgaaaggtgagaaatggatcgcaacgcagtgggtgagaagaggtacttataaggaattc.

[0060] The amino acid sequence of the BaP4H mutant is (SEQ ID NO. 3): MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSAEECDELIELSKSKLARSKVGSSTDVNDIRTSSGAFLDDNELTAKIEKRISSIMNVPASHGEGLHILNYEVDQQYKAHYDYFAEHSRSAANNRISTLVMYLNDVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYKEF.

[0061] (2) When the mutation site is D50A+D129K:

[0062] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO.4): atgacaaacaacaatcaaataggtgaaaataaggaacaaactatttttgatcataaaggaaatgtaattaagacagaggatagagaaatacaaattatttcaaaattcgaagaacctcttattgtcgtgttaggaaatgtattaagtgcagaagagtgtgatgaattaattgaattgtctaaaagtaaattagcacgttcaaaagttggttcatcacgtgatgtaaatgatattcgaacgagtagtggtgcatttttggacgataatgaacttacggcgaagattgaaaaacggatttcatctatcatgaatgttcctgcgtcgcatggagaaggattacacattttaaattatgaagtggatcaacaatataaagcgcattataaatattttgcggaacatagtagatccgctgctaataatcgtattagtacgcttgttatgtacttaaatgatgtcgaagaaggcggagaaacgttctttccgaaattaaatctttctgtgcaccctagaaagggaatggcagtatactttgagtatttctatcaagaccaatcattaaacgagcttacgttacacggaggggcacctgtaacgaaaggtgagaaatggatcgcaacgcagtgggtgagaagaggtacttataaggaattc。

[0063] The amino acid sequence of the BaP4H mutant is (SEQ ID NO.5): MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSAEECDELIELSKSKLARSKVGSSRDVNDIRTSSGAFLDDNELTAKIEKRISSIMNVPASHGEGLHILNYEVDQQYKAHYKYFAEHSRSAANNRISTLVMYLNDVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYKEF。

[0064] (3) When the mutation site is D50A + D152G:

[0065] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO.6): atgacaaacaacaatcaaataggtgaaaataaggaacaaactatttttgatcataaaggaaatgtaattaagacagaggatagagaaatacaaattatttcaaaattcgaagaacctcttattgtcgtgttaggaaatgtattaagtgcagaagagtgtgatgaattaattgaattgtctaaaagtaaattagcacgttcaaaagttggttcatcacgtgatgtaaatgatattcgaacgagtagtggtgcatttttggacgataatgaacttacggcgaagattgaaaaacggatttcatctatcatgaatgttcctgcgtcgcatggagaaggattacacattttaaattatgaagtggatcaacaatataaagcgcattatgattattttgcggaacatagtagatccgctgctaataatcgtattagtacgcttgttatgtacttaaatggagtcgaagaaggcggagaaacgttctttccgaaattaaatctttctgtgcaccctagaaagggaatggcagtatactttgagtatttctatcaagaccaatcattaaacgagcttacgttacacggaggggcacctgtaacgaaaggtgagaaatggatcgcaacgcagtgggtgagaagaggtacttataaggaattc.

[0066] The amino acid sequence of the BaP4H mutant is (SEQ ID NO.7): MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSAEECDELIELSKSKLARSKVGSSRDVNDIRTSSGAFLDDNELTAKIEKRISSIMNVPASHGEGLHILNYEVDQQYKAHYDYFAEHSRSAANNRISTLVMYLNGVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYKEF.

[0067] (4) When the mutation site is D50A+H134T:

[0068] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO.8): atgacaaacaacaatcaaataggtgaaaataaggaacaaactatttttgatcataaaggaaatgtaattaagacagaggatagagaaatacaaattatttcaaaattcgaagaacctcttattgtcgtgttaggaaatgtattaagtgcagaagagtgtgatgaattaattgaattgtctaaaagtaaattagcacgttcaaaagttggttcatcacgtgatgtaaatgatattcgaacgagtagtggtgcatttttggacgataatgaacttacggcgaagattgaaaaacggatttcatctatcatgaatgttcctgcgtcgcatggagaaggattacacattttaaattatgaagtggatcaacaatataaagcgcattatgattattttgcggaaactagtagatccgctgctaataatcgtattagtacgcttgttatgtacttaaatgatgtcgaagaaggcggagaaacgttctttccgaaattaaatctttctgtgcaccctagaaagggaatggcagtatactttgagtatttctatcaagaccaatcattaaacgagcttacgttacacggaggggcacctgtaacgaaaggtgagaaatggatcgcaacgcagtgggtgagaagaggtacttataaggaattc。

[0069] The amino acid sequence of the BaP4H mutant is (SEQ ID NO.9): MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSAEECDELIELSKSKLARSKVGSSRDVNDIRTSSGAFLDDNELTAKIEKRISSIMNVPASHGEGLHILNYEVDQQYKAHYDYFAETSRSAANNRISTLVMYLNDVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYKEF。

[0070] (5) When the mutation site is D50A+H114D:

[0071] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO.10): atgacaaacaacaatcaaataggtgaaaataaggaacaaactatttttgatcataaaggaaatgtaattaagacagaggatagagaaatacaaattatttcaaaattcgaagaacctcttattgtcgtgttaggaaatgtattaagtgcagaagagtgtgatgaattaattgaattgtctaaaagtaaattagcacgttcaaaagttggttcatcacgtgatgtaaatgatattcgaacgagtagtggtgcatttttggacgataatgaacttacggcgaagattgaaaaacggatttcatctatcatgaatgttcctgcgtcgcatggagaaggattagacattttaaattatgaagtggatcaacaatataaagcgcattatgattattttgcggaacatagtagatccgctgctaataatcgtattagtacgcttgttatgtacttaaatgatgtcgaagaaggcggagaaacgttctttccgaaattaaatctttctgtgcaccctagaaagggaatggcagtatactttgagtatttctatcaagaccaatcattaaacgagcttacgttacacggaggggcacctgtaacgaaaggtgagaaatggatcgcaacgcagtgggtgagaagaggtacttataaggaattc.

[0072] The amino acid sequence of the BaP4H mutant is (SEQ ID NO.11): MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSAEECDELIELSKSKLARSKVGSSRDVNDIRTSSGAFLDDNELTAKIEKRISSIMNVPASHGEGLDILNYEVDQQYKAHYDYFAEHSRSAANNRISTLVMYLNDVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYKEF.

[0073] (6) When the mutation site is D50A+R142E:

[0074] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO.12): atgacaaacaacaatcaaataggtgaaaataaggaacaaactatttttgatcataaaggaaatgtaattaagacagaggatagagaaatacaaattatttcaaaattcgaagaacctcttattgtcgtgttaggaaatgtattaagtgcagaagagtgtgatgaattaattgaattgtctaaaagtaaattagcacgttcaaaagttggttcatcacgtgatgtaaatgatattcgaacgagtagtggtgcatttttggacgataatgaacttacggcgaagattgaaaaacggatttcatctatcatgaatgttcctgcgtcgcatggagaaggattacacattttaaattatgaagtggatcaacaatataaagcgcattatgattattttgcggaacatagtagatccgctgctaataatgagattagtacgcttgttatgtacttaaatgatgtcgaagaaggcggagaaacgttctttccgaaattaaatctttctgtgcaccctagaaagggaatggcagtatactttgagtatttctatcaagaccaatcattaaacgagcttacgttacacggaggggcacctgtaacgaaaggtgagaaatggatcgcaacgcagtgggtgagaagaggtacttataaggaattc。

[0075] The amino acid sequence of the BaP4H mutant is (SEQ ID NO.13): MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSAEECDELIELSKSKLARSKVGSSRDVNDIRTSSGAFLDDNELTAKIEKRISSIMNVPASHGEGLHILNYEVDQQYKAHYDYFAEHSRSAANNEISTLVMYLNDVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYKEF。

[0076] (7) When the mutation site is D50A + S81L:

[0077] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO.14): atgacaaacaacaatcaaataggtgaaaataaggaacaaactatttttgatcataaaggaaatgtaattaagacagaggatagagaaatacaaattatttcaaaattcgaagaacctcttattgtcgtgttaggaaatgtattaagtgcagaagagtgtgatgaattaattgaattgtctaaaagtaaattagcacgttcaaaagttggttcatcacgtgatgtaaatgatattcgaacgcttagtggtgcatttttggacgataatgaacttacggcgaagattgaaaaacggatttcatctatcatgaatgttcctgcgtcgcatggagaaggattacacattttaaattatgaagtggatcaacaatataa

[0078] agcgcattatgattattttgcggaacatagtagatccgctgctaataatcgtattagtacgcttgttatgtacttaaatgatgtcgaagaaggc

[0079] ggagaaacgttctttccgaaattaaatctttctgtgcaccctagaaagggaatggcagtatactttgagtatttctatcaagaccaatcattaa

[0080] acgagcttacgttacacggaggggcacctgtaacgaaaggtgagaaatggatcgcaacgcagtgggtgagaagaggtacttataaggaattc.

[0081] The amino acid sequence of the BaP4H mutant is (SEQ ID NO.15):

[0082] MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSAEECDELIELSK

[0083] SKLARSKVGSSRDVNDIRTLSGAFLDDNELTAKIEKRISSIMNVPASHGEGLHILNYEVD

[0084] QQYKAHYDYFAEHSRSAANNRISTLVMYLNDVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYKEF.

[0085] 4. The mutants were constructed and plasmids were extracted according to the method of Example 1, and co-transfected with pET28a-SEQ9 into Rosetta competent cells for protein expression and purification.

[0086] SDS-PAGE verification of recombinant collagen expression ( Figure 2 ), Western Blot-strep verification of the expression of BaP4H mutants ( Figure 3 ).

[0087] Example 3: Determination of hydroxylation rate of recombinant collagen

[0088] The hydroxylation rate was determined using the Solebol hydroxyproline (HYP) content detection kit.

[0089] Preheat the microplate reader for more than 30 minutes and adjust the wavelength to 560nm. Dilute the standard with ultrapure water to 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, and 0.234μg / mL standard solutions. After dialysis, the protein was uniformly diluted to 0.3mg / mL, and 6M HCl was added at a ratio of 1:1 (V / V). After mixing, the mixture was sealed and kept warm at 110℃ for 8h. After cooling, the pH was adjusted to neutral with NaOH and the volume was fixed. The standard curve and sample tests of the above samples were drawn according to the table:

[0090]

[0091] Calculation of hydroxylation rate: first calculate the proportion of proline in the protein sequence B (i.e. the proportion of proline in the amino acids), then calculate the protein concentration C after dilution (i.e. the protein is diluted by the extract and neutralization solution, generally 3 times); then substitute x from the previous step into the final formula: hydroxylation rate (%) = x / (B×C)×100%.

[0092] The experimental results showed that the collagen co-expressed with the BaP4H mutant was hydroxylated, and the hydroxylation rates of C5 and C11 were increased by 2.35 times and 1.66 times respectively compared with the wild type.

[0093] Example 4: Determination of stability of hydroxylated recombinant collagen

[0094] The stability test of recombinant collagen was determined using a circular dichroism spectrometer. The dialyzed protein samples were diluted to the same concentration for later use. The dialysate was mixed with water in a ratio of 1:2 (V / V) as a blank group. The room temperature CD baseline was measured at 190-260nm under the "SpectraMeasurement" program; the protein was mixed with water in a ratio of 1:2 (V / V) as a test. The dialysate was mixed with water in a ratio of 1:15 (V / V) as a blank group. In the "Temperature Interval Measurement" program, the variable temperature CD baseline was measured at 190-260nm, 25-95℃, and a step size of 1℃. The remaining samples were measured in the same way. The room temperature CD results showed that the above mutants all had triple helix characteristic peaks, and the variable temperature CD results ( Figure 4 ) showed that the Tm value of SEQ9 after hydroxylation by the BaP4H mutant was higher than that of SEQ9 hydroxylated by the wild-type BaP4H, preferably by 3°C, indicating that the efficient hydroxylation of recombinant collagen by the BaP4H mutant of the present invention enhanced the stability of collagen.

Claims

1. A BaP4H mutant, characterized in that: The mutant at least includes a mutation site in which the aspartic acid at position 50 of the polypeptide shown in SEQ ID NO.1 is mutated to alanine.

2. The BaP4H mutant according to claim 1, characterized in that The mutation sites are: D50A+R73T, D50A+D129K, D50A+D152G, D50A+H134T, D50A+H114D, D50A+R142E or D50A+S81L.

3. The BaP4H mutant according to claim 2, characterized in that When the mutation site is D50A+R73T, the amino acid sequence of the BaP4H mutant is shown in SEQ ID NO.3; or, when the mutation site is D50A+D129K, the amino acid sequence of the BaP4H mutant is shown in SEQ ID NO.5; or, when the mutation site is D50A+D152G, the amino acid sequence of the BaP4H mutant is shown in SEQ ID NO.7; or, when the mutation site is D50A+H134T, the amino acid sequence of the BaP4H mutant is shown in SEQ ID NO.9; or, when the mutation site is D50A+H114D, the amino acid sequence of the BaP4H mutant is shown in SEQ ID NO.11; or, when the mutation site is D50A+R142E, the amino acid sequence of the BaP4H mutant is shown in SEQ ID NO.13; or, when the mutation site is D50A+S81L, the amino acid sequence of the BaP4H mutant is shown in SEQ ID NO.

15.

4. A gene encoding the BaP4H mutant according to claim 3, characterized in that: The nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.3 is shown in SEQ ID NO.2; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.5 is shown in SEQ ID NO.4; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.7 is shown in SEQ ID NO.6; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.9 is shown in SEQ ID NO.8; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.11 is shown in SEQ ID NO.10; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.13 is shown in SEQ ID NO.12; or, the nucleotide sequence of the gene encoding the BaP4H mutant whose amino acid sequence is shown in SEQ ID NO.15 is shown in SEQ ID NO.

14.

5. A recombinant expression vector, characterized in that: Contains the coding gene as described in claim 4.

6. The recombinant expression vector according to claim 5, characterized in that The vector is pGRO 7, and a strep-tag is added to pGRO 7.

7. A recombinant cell, characterized in that The recombinant cell contains the recombinant expression vector described in claim 5, or the exogenous coding gene described in claim 4 is integrated into its chromosome.

8. An enzyme preparation, characterized in that The method comprises the BaP4H mutant as claimed in any one of claims 1 to 3.

9. Use of the BaP4H mutant according to any one of claims 1 to 3, or the encoding gene according to claim 4, or the recombinant vector according to claim 5, or the recombinant cell according to claim 7, or the enzyme preparation according to claim 8 in catalyzing the proline hydroxylation of recombinant collagen.

10. The use according to claim 9, characterized in that The BaP4H mutant is co-expressed with recombinant collagen to catalyze the hydroxylation of proline in the recombinant collagen, comprising the following steps: i) constructing the coding gene of the BaP4H mutant into the pGRO7-strep plasmid and the coding gene of the recombinant collagen into the pET28a plasmid, mixing them and co-transforming them into Escherichia coli Rosetta competent cells, applying them on double-antibody LB solid culture medium and culturing them overnight; ii) picking up a single colony grown on the plate and culturing it in liquid SOC dual-antibody culture medium as a seed solution; iii) The seed solution was inoculated into the double-resistance LB liquid culture medium, and then cultured for 1 hour, and arabinose with a final concentration of 2 mg / mL was added, and IPTG with a final concentration of 1 mM was added to induce the culture.

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