Bap4h mutant, preparation method therefor, and use thereof

Abstract

The present invention provides a BaP4H mutant, a preparation method therefor, and a use thereof. The BaP4H mutant is obtained by performing mutation on the amino acid sequence shown in SEQ ID NO.1. The present invention further provides a recombinant expression vector expressing the BaP4H mutant, a recombinant cell, a preparation method therefor, and a use thereof.

Description

A BaP4H mutant, its preparation method and application Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and in particular relates to a BaP4H mutant, its preparation method and application. Background Technology

[0002] Proline hydroxylation is the most common post-translational modification in collagen. The resulting 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, affecting its binding to integrins. Prolyl 4-hydroxylases (P4Hs) are ferrous ion- and 2-ketoglutarate-dependent oxidases that catalyze Hyp formation. P4H (BaP4H) from Bacillus anthracis can modify collagen-like proline-rich peptides.

[0003] Collagen's triple helix structure contains abundant, highly repeating tripeptide motifs: Gly-XY, where the X and Y positions are often occupied by proline and hydroxyproline, respectively. In vivo, the proline at the X position needs to be hydroxylated to generate 4-hydroxyproline, forming a stable collagen triple helix structure.

[0004] Currently, some progress has been made in the co-expression of collagen and P4Hs genes in yeast to achieve the production of hydroxylated collagen in industrial expression systems. However, wild-type P4Hs suffers from low expression levels, low hydroxylation efficiency of collagen, and unimproved stability of the recombinant collagen after hydroxylation. Summary of the Invention

[0005] To address the above problems, this invention provides a BaP4H mutant. Experimental results show that the mutant can efficiently hydroxylate recombinant collagen and enhance its stability.

[0006] The first objective of this invention is to provide a BaP4H mutant, which is a mutation in the amino acid sequence shown in SEQ ID NO.1, including deletions and substitutions.

[0007] Preferably, the modification site and the amino acids before and after the mutation are selected from any one of the following:

[0008] The following groups are included: N3S, N4S, N5S, I7M, G8N, E9R, N10E, K11I, E12K, T14E, I15R, D17A, H18D, K19D, G20E, N21S, I23H, K24T, T25A, E26R, D27 (deleted), R28K, E29A, I30F, I32 (deleted), I33 (deleted), S34E, K35G, E37S, E38N, L40M, I41P, L44Y, G45Y, N46A, L48S, S49E, D50A, E51W, E52 (deleted), D54A, E55V, L56N, I57K, E58W, L59Q Missing S60A, K61, S62E, K63L, L64T, A65N, R66P, S67A, K68A, G70T, S71L, S72, R73T, D74A, N76S, D77P, I78A, R79A, R79T, R79S, R79L, S81L, S82, G83N, A84T, L86K, D87K, D88V, N89E, E90S, L91V, T92M, A93D, K94L, I95V, E96M, K97Q, R98S, I99D, S100, S101A, I102A, N104Q, A107T, S108K H109L, G110S, E111M, G112A, L113A, H114D, I115E, L116 missing, 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 missing, A196C, T199S, K200V, G201M, E202Q, I205V, A206K, T207S, R211A, R212Y, G213P, T214Q, and E217N, and combinations thereof.

[0009] More preferably, the mutant wherein each modification is independently a substitution or deletion, wherein the variant has at least 65%, such as 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.

[0010] A second objective of this 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.

[0011] Preferably, the nucleic acid molecule comprises a nucleotide sequence encoding the BaP4H mutant or its complementary sequence.

[0012] Preferably, the recombinant expression vector contains the aforementioned nucleic acid molecule. The expression vector comprises any nucleic acid molecule derived from any source and capable of genomic integration or autonomous replication (e.g., plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules), containing one or more nucleic acid molecules that are operatively linked. The vector may include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the host cell genome.

[0013] Preferably, the recombinant cell contains the recombinant expression vector, or the exogenous nucleic acid molecule is integrated into its chromosome.

[0014] A third objective of this invention is to provide an enzyme preparation comprising the BaP4H mutant described above.

[0015] A fourth objective of this invention is to provide the application of the aforementioned BaP4H mutant, nucleic acid molecule, recombinant expression vector, recombinant cell, and enzyme preparation containing the BaP4H mutant in the catalytic hydroxylation of recombinant collagen proline.

[0016] Preferably, the application involves co-expressing the BaP4H mutant with recombinant collagen to catalyze the hydroxylation of proline in the recombinant collagen, comprising the following steps:

[0017] i) The encoding gene of the BaP4H mutant was constructed into the pGRO-strep plasmid and the encoding gene of recombinant collagen was constructed into the pET28a plasmid. The mixture was then co-transformed into Escherichia coli Rosetta competent cells and plated on double-antibiotic LB solid medium for overnight culture.

[0018] ii) Pick the single colonies that grow on the plate and transfer them to liquid SOC double-antibiotic medium. Incubate overnight at 37°C and 220 rpm to use as seed culture.

[0019] iii) Inoculate the seed culture into double-antibiotic LB liquid medium and add arabinose, then add 1mM IPTG to induce culture.

[0020] More preferably, after inoculating the seed culture into double-antibiotic LB liquid medium and culturing for 1 hour, arabinose with a final concentration of 2 mg / ml is added.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a BaP4H mutant that can efficiently hydroxylate recombinant collagen and further enhance the stability of recombinant collagen; this invention also optimizes the conditions for recombinant cell culture. Attached Figure Description

[0023] Figure 1 shows the effect of different co-expression conditions of BaP4H and recombinant collagen on the structure of recombinant collagen.

[0024] Figure 2 shows the SDS-PAGE validation results of recombinant collagen after co-expression of BaP4H mutant and recombinant collagen.

[0025] Figure 3 shows the Western Blot-strep validation results of the BaP4H mutant after co-expression with recombinant collagen.

[0026] Figure 4 shows the stability test results of recombinant collagen hydroxylated by BaP4H mutant. Detailed Implementation

[0027] This invention does not impose any particular limitation on the preparation method of the recombinant vector; any conventional recombinant vector preparation method in the art can be used. In this invention, the gene can be synthesized by a biotechnology company. This invention does not impose any particular limitation on the separation and purification method; any conventional protein separation and purification method in the art can be used; preferred technical solutions are described in the embodiments.

[0028] This invention characterizes the structure of collagen using circular dichroism (CD), a commonly used spectroscopic method in the field. CD is used to determine the structure of compounds with chiral structures that produce differential absorption between left and right rotations, and is mainly used to determine the asymmetry of molecular structures. Most biological macromolecules contain chiral groups and structures; therefore, CD is often used to measure and observe changes in the structure and conformation of biological macromolecules. The CD characteristic of the triple helix structure of collagen generally shows a positive absorption peak near 221 nm and a negative absorption peak near 195 nm (industry standard YY / T 1849—2022). The positions of the absorption peaks shift with changes in the amino acid sequence and length. The thermal stability of proteins is generally expressed using the melting temperature (Tm), which is the temperature at which a protein unfolds to 50%. For collagen, this refers to the temperature at which the triple helix structure unwinds, forming individual single chains, reaching 50% unwinding. Therefore, CD spectroscopy can be used to study the helical structure of collagen and its thermal denaturation process.

[0029] The technical solutions provided by the present invention will be 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.

[0030] Example 1: Optimization of co-expression conditions of BaP4H and recombinant collagen SEQ9

[0031] pGRO-strep-BaP4H and pET28a-SEQ9 plasmids were constructed and electroporated into Rosetta competent cells, respectively, and plated on solid LB agar plates (Cm). + The final concentration was 34 μg / mL, and the culture was inverted and incubated overnight at 37°C. A single wild-type BaP4H colony grown on the plate was picked and transferred to 4 mL of liquid SOC medium (Chloramphenicol final concentration 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-transformed into Rosetta competent cells and plated on double-antibiotic LB solid medium (Chloramphenicol final concentration 34 μg / mL, Kanamic final concentration 50 μg / mL) for overnight incubation.

[0032] Single colonies grown on the plate were picked and transferred to 10 mL of double-antibiotic liquid LB medium (Chloramphenicol final concentration 34 μg / mL, Kanamic final concentration 50 μg / mL), and incubated overnight at 37°C. 10 mL of the bacterial culture was then transferred to 100 mL of double-antibiotic liquid LB medium and incubated overnight at 37°C. 10 mL of the overnight activated liquid culture was then transferred to 1 L of liquid LB medium at a 1% inoculum rate, divided into 7 groups, and induced to express the bacteria according to the culture conditions listed in tables 1-7.

[0033] Cultivars were collected by centrifugation at 7000 rpm and 4°C for 30 min. The cells were reconstituted with Lysis Buffer at a ratio of 1:8 (w / v) and homogenized using an autoclave. Whole cells were then centrifuged at 18000 rpm for 45 min at 4°C. The supernatant was filtered through a 0.45 μm filter. 1 mL of Ni-beads was added per 1 L of bacterial culture, and the protein was purified by affinity chromatography. After purification, the protein was dialyzed against potassium phosphate buffer. The dialyzed protein was then analyzed by circular dichroism spectroscopy (CD).

[0034] The experimental results are shown in Figure 1. Adding 2 mg / mL of arabinose 1 hour after amplification yielded the best results, and subsequent co-expression was performed under these conditions.

[0035] Example 2: Co-expression of BaP4H mutant and recombinant collagen (SEQ9)

[0036] The mutant constructed in this invention is obtained by mutating wild-type BaP4H, and the amino acid sequence of wild-type BaP4H is as follows:

[0037] The mutants constructed in this invention and their numbering are shown in the table below:

[0038] The mutants were constructed and plasmids were extracted according to the method in Example 1, and co-transformed with pET28a-SEQ9 into Rosetta competent cells for protein expression and purification.

[0039] SDS-PAGE was used to verify the expression of recombinant collagen (Figure 2), and Western Blot-strep was used to verify the expression of BaP4H mutant (Figure 3).

[0040] Example 3: Determination of hydroxylation rate of recombinant collagen

[0041] The hydroxylation rate was determined using the Solarbio hydroxyproline (HYP) content assay kit.

[0042] Preheat the microplate reader for at least 30 minutes and adjust the wavelength to 560 nm. Dilute the standards with ultrapure water to prepare standard solutions of 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, and 0.234 μg / mL. After dialysis, uniformly dilute the protein to 0.3 mg / mL, add 6M HCl at a 1:1 (V / V) ratio, mix well, seal, and incubate at 110℃ for 8 hours. After cooling, adjust the pH to neutral with NaOH and make up to volume. Plot the standard curve and perform sample testing according to the table above.

[0043] Hydroxylation rate calculation: First, calculate the proportion of proline in the protein sequence B (i.e., the proportion of proline in the amino acid sequence), then calculate the protein concentration C after dilution (i.e., the protein is diluted by the extraction solution and neutralization solution, generally by 3 times); then substitute the x from the previous step into the final formula: Hydroxylation rate (%) = x / (B×C)×100%.

[0044] Experimental results showed that all collagen co-expressed with the BaP4H mutant was hydroxylated, with the hydroxylation rates of C5 and C11 increasing by 2.35 times and 1.66 times compared to the wild type, respectively.

[0045] Example 4: Stability determination of hydroxylated recombinant collagen

[0046] The stability of recombinant collagen was determined using circular dichroism spectroscopy. Dialyzed protein samples were diluted to the same concentration for later use. A 1:2 (v / v) mixture of dialysate and water was used as a blank control, and the CD baseline was measured at 190-260 nm using the "Spectra Measurement" program. A 1:2 (v / v) mixture of protein and water was used for testing. A 1:15 (v / v) mixture of dialysate and water was used as a blank control, and the CD baseline was measured at 190-260 nm, 25-95 °C, with a step size of 1 °C using the "Temperature Interval Measurement" program. The remaining samples were measured in the same manner. Room temperature CD results showed that all mutants exhibited triple-helix characteristic peaks. Variable temperature CD results (Figure 4) showed that the Tm value of SEQ9 hydroxylated by the BaP4H mutant was significantly increased compared to that hydroxylated by wild-type BaP4H, preferably by 3 °C, indicating that the efficient hydroxylation of recombinant collagen by the BaP4H mutant of this invention enhances collagen stability.

Claims

1. A BaP4H mutant, wherein the mutant is capable of efficiently hydroxylating recombinant collagen and comprises a modification at one or more positions corresponding to positions 3, 4, 5, 7, 8, 9, 10, 11, 12, 14, 15, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 37, 38, 40, 41, 44, 45, 46, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 104, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 119, 121, 125, 126, 127, 129, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 150, 151, 152, 154, 155, 156, 157, 158, 159, 163, 164, 165, 166, 167, 169, 171, 172, 173, 175, 179, 184, 186, 187, 188, 189, 190, 191, 192, 193, 194, 196, 199, 200, 201, 202, 205, 206, 207, 211, 212, 213, 214, and 217 of the polypeptide of SEQ ID NO. 1, 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 to the mature polypeptide of SEQ ID NO.

1.

2. The variant of claim 1, wherein the alteration is a substitution, wherein substitution of the naturally occurring amino acid residue at a position with a different amino acid residue results in a BaP4H mutant that, when co-expressed with recombinant collagen, is effective to increase the hydroxylation rate and stability thereof.

3. The variant of claim 1, wherein the alteration is a deletion, wherein deletion of the naturally occurring amino acid residue at a position with a different amino acid residue results in a BaP4H mutant that, when co-expressed with recombinant collagen, is effective to increase the hydroxylation rate and stability thereof.

4. The BaP4H mutant of claim 1, characterized in that, The modified positions and their pre- and post-mutated amino acids are selected from any one of 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.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the BaP4H mutant as claimed in any one of claims 1, 4 or a complementary sequence thereof.

6. A recombinant expression vector, characterized in that, The nucleic acid molecule as claimed in claim 5.

7. The recombinant expression vector of claim 6, wherein, The vector is pGRO 7, and a strep-tag is added to the pGRO 7.

8. A recombinant cell, characterized in that, The recombinant cell contains the recombinant expression vector as claimed in claim 6, or the nucleic acid molecule as claimed in claim 5 is integrated into the chromosome of the recombinant cell.

9. An enzyme preparation, characterized in that, The BaP4H mutant as claimed in any one of claims 1, 4.

10. Use of the BaP4H mutant as claimed in any one of claims 1, 4, or the nucleic acid molecule as claimed in claim 5, or the recombinant expression vector as claimed in claim 6, or the recombinant cell as claimed in claim 8, or the enzyme preparation as claimed in claim 9 in catalyzing the hydroxylation of recombinant collagen proline.

11. Use according to claim 10, characterized in that, The seed liquid is inoculated into a double-antibiotic LB liquid medium and cultured for 1 h, and then 2 mg / ml of arabinose is added. i) The coding gene of the BaP4H mutant and the coding gene of recombinant collagen are constructed into pGRO7-strep plasmid and pET28a plasmid respectively, and then co-transformed into E. coli Rosetta competent cells, and then inoculated onto double-antibiotic LB solid medium and cultured overnight; ii) Single colonies grown on the plate are picked into liquid SOC double-antibiotic medium for culture as seed liquid; iii) The seed liquid is inoculated into double-antibiotic LB liquid medium and cultured, and then arabinose is added, and then 1 mM IPTG is added for induction.

12. Use according to claim 11, characterized in that, The seed liquid is inoculated into a double-antibiotic LB liquid medium and cultured for 1 h, and then 2 mg / ml of arabinose is added.

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