A BaP4H mutant, its preparation method and application

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a BaP4H mutant, a preparation method thereof, and an application thereof. Background Art

[0002] Proline hydroxylation is the most common post-translational modification in collagen. The resulting product, trans-4-hydroxyproline (Hyp), is crucial for the stability and function of collagen. Studies have shown that the lack of proline hydroxylation in collagen can make the triple helix structure unstable, thereby affecting the binding to integrin. 4-Prolyl hydroxylase (Prolyl 4-hydroxylases, P4Hs) is a class of oxidases that depend on ferrous ions and 2-ketoglutaric acid and can catalyze the formation of Hyp. P4H from Bacillus anthracis (BaP4H) can modify collagen-like proline-rich peptides.

[0003] The triple helix structure of collagen contains abundant highly repetitive tripeptide motifs: Gly-X-Y, where the X and Y positions are often 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.

[0004] Currently, certain progress has been made in the co-expression of collagen genes and P4Hs genes in yeast to produce hydroxylated collagen in an industrial expression system. However, wild-type P4Hs have problems such as low expression levels, low hydroxylation efficiency for collagen, and no improvement in the stability of the hydroxylated recombinant collagen. Summary of the Invention

[0005] 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.

[0006] The first object of the present invention is to provide a BaP4H mutant, which is mutated on the amino acid sequence shown in SEQ ID NO.1 and includes deletions and substitutions.

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

[0008] A 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.

[0009] More preferably, each modification in the mutant is independently a substitution or a deletion, and 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] 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.

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

[0012] Preferably, the recombinant expression vector contains the nucleic acid molecule. The expression vector includes any nucleic acid molecule (such as a 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, which contains a nucleic acid molecule to which one or more nucleic acid molecules have been operably linked. The vector may include, for example, one or more selectable markers, one or more origins of replication (such as prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that promote stable integration of the construct into the genome of the host cell.

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

[0014] The third object of the present invention is to provide an enzyme preparation containing the BaP4H mutant.

[0015] The fourth object of the present 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 catalyzing the proline hydroxylation of recombinant collagen.

[0016] Preferably, the application is to co-express the BaP4H mutant with recombinant collagen to catalyze the hydroxylation of proline in recombinant collagen, including the following steps:

[0017] i) Construct the coding gene of the BaP4H mutant into the pGRO-strep plasmid and the coding gene of recombinant collagen into the pET28a plasmid, mix them, and co-transform them into Escherichia coli Rosetta competent cells, and spread them on a double-antibody LB solid medium for overnight culture;

[0018] ii) Pick the single colonies grown on the plate and transfer them into the liquid SOC medium with double antibiotics. Incubate overnight at 37 °C with shaking at 220 rpm as the seed solution.

[0019] iii) Inoculate the seed solution into the double-antibiotic LB liquid medium for culture and add arabinose, and add IPTG with a final concentration of 1 mM for induction culture.

[0020] More preferably, the seed solution is inoculated into the double-antibiotic LB liquid medium for culture for 1 h, and then 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] 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. Description of the Drawings

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

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

[0025] Figure 3 Showing the Western Blot-strep verification results of BaP4H mutant after co-expression of BaP4H mutant and recombinant collagen.

[0026] Figure 4 Showing the stability determination results of recombinant collagen hydroxylated by BaP4H mutant. Detailed Embodiments

[0027] The present invention has no special limitation on the preparation method of the recombinant vector, and the conventional preparation methods of recombinant vectors in the art can be used. In the present invention, the gene can be obtained by synthesis from a biotechnology company. The present invention has no special limitation on the separation and purification method, and the conventional protein separation and purification methods in the art can be used; the preferred technical solutions are described in the examples.

[0028] The structural characterization of collagen in the present invention is carried out by using circular dichroism (CD), a commonly used method in the art. CD is used to determine the structure of compounds with chiral structures that can produce differential absorption of left and right circularly polarized light, and is mainly a spectroscopic method for determining the molecular structure asymmetry. Generally, biological macromolecules contain chiral groups and structures, so CD is often used to measure and observe the structural and conformational changes of biological macromolecules. The CD characteristics of the collagen triple helix structure generally have a positive absorption peak near 221 nm and a negative absorption peak near 195 nm (industry standard YY / T 1849—2022). The position of the absorption peak will shift with changes in the amino acid sequence and length. The thermal stability of proteins is generally expressed by the melting temperature (Tm), that is, the temperature at which the protein unfolds by 50%. For collagen, it refers to the temperature at which the triple helix structure unwinds and forms single strands respectively, and when the triple helix unwinds to 50%. Therefore, the CD spectrum can be used to study the collagen helix structure and its thermal denaturation process.

[0029] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

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

[0031] Construct pGRO-strep-BaP4H plasmid and pET28a-SEQ9 plasmid respectively, and electrotransform them into Rosetta competent cells, and coat them on solid LB plates (Cm + with a final concentration of 34 μg / mL), and incubate them overnight at 37°C in an inverted position. Pick the wild-type BaP4H single colonies grown on the plate into 4 mL of liquid SOC medium (with a final concentration of Chloramphenicol of 34 μg / mL), and culture them overnight at 37°C to extract the plasmid. Extract the pET28a-SEQ9 plasmid in the same way. Co-transform pGRO-strep-BaP4H and pET28a-SEQ9 into Rosetta competent cells, and coat them on double-antibody LB solid medium (with a final concentration of Chloramphenicol of 34 μg / mL and a final concentration of Kanamic of 50 μg / mL) and culture them overnight.

[0032] Pick a single colony grown on the plate and transfer it to 10 mL of double-antibiotic liquid LB medium (final concentration of Chloramphenicol is 34 μg / mL, final concentration of Kanamic is 50 μg / mL), and culture it overnight at 37°C. Transfer 10 mL of the bacterial solution to 100 mL of double-antibiotic liquid LB medium and culture it overnight at 37°C. Take 10 mL each of the overnight-activated liquid bacterial strains and transfer them to 1 L of liquid LB medium according to an inoculation amount of 1%, and divide them into 7 groups, and induce expression according to the culture conditions No. 1-7 in the table respectively:

[0033]

[0034] Centrifuge at 7000 rmp for 30 min at 4°C to harvest the bacteria. Resuspend the bacteria by adding Lysis Buffer according to the ratio of bacterial mass: Lysis Buffer = 1:8 (W / V), and break the cells with a high-pressure homogenizer. Centrifuge the whole bacteria at 18000 rpm for 45 min at 4°C, filter the supernatant through a 0.45 μm filter, add 1 mL of Ni-beads per 1 L of bacterial solution, purify the protein by affinity chromatography, and dialyze it with potassium phosphate buffer after purification. The protein after dialysis is measured by CD using a circular dichroism spectrometer.

[0035] The experimental results are as Figure 1 shown. Adding 2 mg / mL of arabinose 1 h after amplification has the best effect, and subsequent co-expression is carried out under this condition.

[0036] Example 2 Co-expression of BaP4H mutant and recombinant collagen Recombinant collagen SEQ9

[0037] The mutant constructed in the present invention is a mutation of wild-type BaP4H, and the amino acid sequence of the wild-type BaP4H is as follows:

[0038] MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSDEECDELIELSKSKLARSKVGSSRDVNDIRTSSGAFLDDNELTAKIEKRISSIMNVPASHGEGLHILNYEVDQQYKAHYDYFAEHSRSAANNRISTLVMYLNDVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYKEF (SEQ ID NO.1).

[0039] The mutants constructed in the present invention and their numbers are shown in the following table:

[0040] 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

[0041] 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.

[0042] SDS-PAGE verification of recombinant collagen expression ( Figure 2 ), Western Blot-strep verification of the expression of BaP4H mutants ( Figure 3 ).

[0043] Example 3 Determination of hydroxylation rate of recombinant collagen

[0044] The hydroxylation rate was determined using the Solebol hydroxyproline (HYP) content detection kit.

[0045] 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:

[0046]

[0047] 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%.

[0048] 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.

[0049] Example 4 Stability determination of hydroxylated recombinant collagen

[0050] The stability test of recombinant collagen was determined using a circular dichroism spectrometer. The dialyzed protein sample was diluted to the same concentration for standby. The dialysis solution and water were mixed at a ratio of 1:2 (V / V) as the blank group to measure the normal temperature CD baseline at 190 - 260 nm under the "Spectra Measurement" program; the protein and water were mixed at a ratio of 1:2 (V / V) for testing. The dialysis solution and water were mixed at a ratio of 1:15 (V / V) as the blank group in the "Temperature Interval Measurement" program, and the temperature-variable CD baseline was measured at 190 - 260 nm, 25 - 95 °C, with a step size of 1 °C. The remaining samples were measured in the same way. The normal temperature CD results showed that all the above mutants had triple helix characteristic peaks, and the temperature-variable CD results ( Figure 4 ) showed that for SEQ9 hydroxylated by the BaP4H mutant, its Tm value was increased compared with that hydroxylated by the wild-type BaP4H, preferably increased by 3 °C, indicating that the efficient hydroxylation of the 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: D17A, D17A+S67A, D17A+N21S, D17A+I102A or D17A+R211A.

2. An enzyme preparation, characterized in that Comprising the BaP4H mutant as claimed in claim 1.

3. Use of the BaP4H mutant according to claim 1 or the enzyme preparation according to claim 2 in catalyzing the proline hydroxylation of recombinant collagen.

4. The use according to claim 3, 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, and applying them on double-antibody LB solid medium for overnight culture; ii) Pick up 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 double-resistance LB liquid medium and arabinose was added, and IPTG with a final concentration of 1 mM was added to induce the culture.

5. The use according to claim 4, characterized in that: The seed solution was inoculated into the double-antibody LB liquid medium and then cultured for 1 hour and then arabinose was added at a final concentration of 2 mg / mL.

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