A BaP4H mutant and its application

Abstract

The present invention provides a BaP4H mutant and its application, belonging to the field of enzyme engineering technology. The BaP4H mutant is mutated in the amino acid sequence shown in SEQ ID NO. 1. The present invention also provides a recombinant expression vector and recombinant cell expressing the BaP4H mutant, as well as a preparation method and application. The BaP4H mutant provided by the present invention efficiently hydroxylates proline in recombinant collagen and enhances the stability of the recombinant collagen.

Description

[0001] This application is a divisional application with an application date of July 31, 2024, application number 202411037202.3, and invention name “A BaP4H mutant, its preparation method and application”. 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 prevalent post-translational modification in collagen. The resulting product, trans-4-hydroxyproline (Hyp), is crucial for collagen stability and function. Studies have shown that a lack of proline hydroxylation in collagen destabilizes the triple helix structure, thereby impairing integrin binding. Proline 4-hydroxylases (P4Hs) are a class of oxidases that rely on ferrous ions and 2-oxoglutarate and catalyze the formation of Hyp. P4H from Bacillus anthracis (BaP4H) can modify collagen-like proline-rich peptides.

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

[0005] Currently, progress has been made in the co-expression of collagen and P4Hs genes in yeast, enabling the production of hydroxylated collagen in industrial expression systems. However, wild-type P4Hs suffer from low expression levels, low collagen hydroxylation efficiency, and poor stability of the recombinant collagen after hydroxylation. Summary of the Invention

[0006] 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 mutant 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] 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 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 for 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 comprises any nucleic acid molecule (such as 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 genomic integration or autonomous replication, and it comprises the nucleic acid molecule of one or more nucleic acid molecules that are operably connected. The carrier may comprise, for example, one or more selectable markers, one or more replication origins (such as protokaryotic and eukaryotic origins), at least one multiple cloning site and / or promotes the stable integration of the construct into the element 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 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. Shown in NO.15.

[0021] Preferably, the present invention provides the coding gene of the above-mentioned BaP4H mutant, specifically: the nucleotide sequence of the coding gene of 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 coding gene of 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 coding gene of 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 coding gene of 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 coding gene of 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 coding gene of 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 coding gene of 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 comprising 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, and smearing them on double-antibody LB solid medium for overnight culture;

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

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

[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 the recombinant collagen; the present invention also optimizes the conditions for recombinant cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The results show 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 mutants and recombinant collagen.

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

[0035] Figure 4 Shown are the results of stability assays 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; conventional methods for preparing recombinant vectors in the art can be employed. 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; conventional protein separation and purification methods in the art can be employed. Preferred technical solutions are described in the Examples.

[0037] The present invention characterizes the structure of collagen using circular dichroism (CD), a spectroscopic method commonly used in the field. CD is used to determine the structure of compounds with chiral structures that produce differential absorption of left- and right-handed optical rotation, and is primarily used to measure molecular structural asymmetry. Biomacromolecules generally contain chiral groups and structures, and therefore CD is often used to measure and observe structural and conformational changes in biomacromolecules. The CD characteristic of collagen's triple helix structure is generally characterized by a positive absorption peak near 221nm and a negative absorption peak near 195nm (industry standard YY / T1849-2022). The positions of the absorption peaks shift with changes in amino acid sequence and length. The thermal stability of proteins is generally measured using the melting temperature (Tm), which is the temperature at which the protein unfolds by 50%. For collagen, this refers to the temperature at which the triple helix unwinds, forming single strands, and the triple helix unwinds by 50%. Therefore, CD spectroscopy 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 with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

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

[0040] pGRO-strep-BaP4H plasmid and pET28a-SEQ9 plasmid were constructed and electroporated into Rosetta competent cells respectively, and then plated on solid LB plates (Cm + The plate was inverted and cultured overnight at 37°C. A single wild-type BaP4H colony grown on the plate was transferred to 4 mL of liquid SOC medium (final chloramphenicol concentration of 34 μg / mL) and cultured overnight at 37°C to extract the plasmid. The pET28a-SEQ9 plasmid was extracted using the same method. pGRO-strep-BaP4H and pET28a-SEQ9 were co-transfected into Rosetta competent cells and plated onto double-antibody LB solid medium (final chloramphenicol concentration of 34 μg / mL, final Kanamic concentration of 50 μg / mL) for overnight culture.

[0041] Single colonies grown on the plates were transferred to 10 mL of double-antibody liquid LB medium (final concentration of Chloramphenicol: 34 μg / mL, final concentration of Kanamic: 50 μg / mL) and cultured overnight at 37°C. 10 mL of the bacterial suspension was transferred to 100 mL of double-antibody liquid LB medium and cultured overnight at 37°C. 10 mL of each overnight activated liquid culture was transferred to 1 L of liquid LB medium at a 1% inoculum size. The cells were divided into 7 groups and induced to express according to the culture conditions 1-7 in the table:

[0042]

[0043] Harvest the cells by centrifugation at 7000 rpm for 30 min at 4°C. Redissolve the cells by adding Lysis Buffer at a ratio of 1:8 (w / v) and disrupt the cells using a high-pressure homogenizer. Disrupt the cells by centrifugation at 18000 rpm for 45 min at 4°C. Filter the supernatant through a 0.45 μm filter and add 1 mL of Ni-beads per liter of culture. Purify the protein by affinity chromatography and dialyze against potassium phosphate buffer. Analyze the dialyzed protein by CD spectrometry.

[0044] The experimental results are as follows Figure 1 As shown in the figure, 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 SEQ9

[0046] 1. The mutant constructed in the present invention is a mutation of the wild-type BaP4H, the amino acid sequence of which 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 sites are 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。<00001​​​​(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 BaP4H mutant expression ( Figure 3 ).

[0087] Example 3: Determination of the 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, seal and incubate at 110℃ for 8h. After cooling, adjust the pH to neutral with NaOH and make up to volume. Draw the standard curve and sample test for the above samples according to the table:

[0090]

[0091] Calculation of hydroxylation rate: First calculate the proline ratio B in the protein sequence (i.e., the proportion of proline in the total amino acids), then calculate the diluted protein concentration C (i.e., the protein is diluted with the extract and neutralization solution, generally 3 times). Substituting x from the previous step, the final formula is: 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: Stability determination of hydroxylated recombinant collagen

[0094] The stability test of recombinant collagen was determined using circular dichroism spectrometer. The dialyzed protein samples were diluted to the same concentration for later use. The dialysate was mixed with water at a ratio of 1:2 (V / V) as a blank group and the room temperature CD baseline was measured at 190-260nm under the "SpectraMeasurement" program; the protein was mixed with water at a ratio of 1:2 (V / V) as a test. The dialysate was mixed with water at a ratio of 1:15 (V / V) as a blank group and the variable temperature CD baseline was measured at 190-260nm, 25-95℃, and a step size of 1℃ in the "Temperature Interval Measurement" program. 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 increased compared with that after hydroxylation by the wild-type BaP4H, preferably increased 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 is mutated based on the polypeptide shown in SEQ ID NO.1, and the specific mutation sites are: D50A+R73T, D50A+D129K, D50A+D152G, D50A+H134T, D50A+H114D, D50A+R142E or D50A+S81L.

2. The BaP4H mutant according to claim 1, wherein 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.

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

14.

4. A recombinant expression vector, characterized in that: Contains the coding gene described in claim 3.

5. The recombinant expression vector according to claim 4, wherein The vector is pGRO 7, and a strep-tag is added to pGRO 7.

6. A recombinant cell, characterized in that The recombinant cell contains the recombinant expression vector described in claim 4, or the exogenous coding gene described in claim 3 is integrated into its chromosome.

7. An enzyme preparation, characterized in that The method comprises the BaP4H mutant according to claim 1 or 2.

8. Use of the BaP4H mutant according to claim 1 or 2, or the encoding gene according to claim 3, or the recombinant expression vector according to claim 4, or the recombinant cell according to claim 6, or the enzyme preparation according to claim 7 in catalyzing the proline hydroxylation of recombinant collagen.

9. The use according to claim 8, 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 BaP4H mutant encoding gene into the pGRO 7-strep plasmid and the recombinant collagen encoding gene into the pET28a plasmid, mixing the mixture and co-transforming into Escherichia coli Rosetta competent cells, and smearing on double-antibody LB solid medium for overnight culture; ii) Pick a single colony grown on the plate and culture it in liquid SOC dual-antibody medium as a seed solution; iii) The seed solution was inoculated into a double-antibody LB liquid culture medium and cultured for 1 hour, followed by the addition of arabinose at a final concentration of 2 mg / mL and the induction of the culture by adding IPTG at a final concentration of 1 mM.

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