Bap4h mutant and use thereof

Abstract

The present invention provides a BaP4H mutant 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 and application thereof

[0001] This application is a divisional application of the application with the application number 202411037202.3 and the application name "A BaP4H mutant, preparation method and application thereof", the application date of which is July 31, 2024. TECHNICAL FIELD

[0002] The present application belongs to the technical field of enzyme engineering, and particularly relates to a BaP4H mutant and application thereof. BACKGROUND

[0003] Proline hydroxylation is the most common post-translational modification in collagen. The product generated thereby, trans-4-hydroxyproline (Hyp), is essential for the stability and function of collagen. It has been shown that the lack of proline hydroxylation in collagen destabilizes the triple helix and impairs binding to integrins. Prolyl 4-hydroxylases (P4Hs) are a class of 2-oxoglutarate-dependent oxygenases that catalyze the formation of Hyp. P4H from Bacillus anthracis (BaP4H) can modify collagen-like peptides rich in proline.

[0004] The triple helical structure of collagen contains a richly repetitive tripeptide motif: Gly-X-Y, where X and Y are often proline and hydroxyproline. In vivo, the proline at the X position needs to be hydroxylated to generate 4-hydroxyproline, forming a stable triple helical structure of collagen.

[0005] At present, the co-expression of collagen genes and P4Hs genes in yeast has made some progress to produce hydroxylated collagen in an industrial expression system. However, wild-type P4Hs have low expression levels, low hydroxylation efficiency for collagen, and the stability of the recombinant collagen after hydroxylation is not improved. SUMMARY

[0006] To solve the above problems, the present application provides a BaP4H mutant, and 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 application is to provide a BaP4H mutant, which is mutated on the amino acid sequence shown in SEQ ID NO. 1, and contains deletions and substitutions.

[0008] Preferably, the modified position and its amino acids before and after 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] A second object of the present application is to provide an expression system of the BaP4H mutant, including but not limited to nucleic acid molecules, recombinant expression vectors, 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., a plasmid, a cosmid, a virus, an autonomously replicating polynucleotide molecule, a bacteriophage, or a linear or circular single- or double-stranded DNA or RNA nucleic acid molecule) derived from any source and capable of genomic integration or autonomous replication, which comprises a nucleic acid molecule that has been operably linked to one or more nucleic acid molecules. The vector can 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 genome of a host cell.

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

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

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

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

[0018] The present application provides a BaP4H mutant, which comprises at least 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 NO. 15.

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

[0022] Preferably, the present application provides a recombinant expression vector containing the above-mentioned coding gene, and the vector is pGRO 7, and a strep-tag is added to the pGRO 7.

[0023] Preferably, the present application provides a recombinant cell containing the above-mentioned recombinant expression vector, or the above-mentioned coding gene is integrated into the chromosome of the recombinant cell.

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

[0025] Preferably, the present application 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 hydroxylation of recombinant collagen proline.

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

[0027] i) construct the encoding gene of the BaP4H mutant into a pGRO7-strep plasmid and the encoding gene of recombinant collagen into a pET28a plasmid, and then co-transform into E. coli Rosetta competent cells, and then inoculate on double-antibiotic LB solid medium for overnight culture;

[0028] ii) pick single colonies growing on the plate into liquid SOC double-antibiotic medium for culture as seed liquid;

[0029] iii) inoculate the seed liquid into double-antibiotic LB liquid medium, and then incubate for 1 h, then add arabinose at a final concentration of 2 mg / mL, and then add IPTG at a final concentration of 1 mM for induction culture.

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

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

[0032] Figure 1 shows the influence of different co-expression conditions of BaP4H and recombinant collagen on the structure of recombinant collagen.

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

[0034] Figure 3 shows the Western Blot-strep verification results of BaP4H mutant after co-expression of BaP4H mutant and recombinant collagen.

[0035] Figure 4 shows the stability determination results of recombinant collagen after hydroxylation by BaP4H mutant. DETAILED DESCRIPTION

[0036] The preparation method of the recombinant vector is not particularly limited in the present application, and the conventional preparation method of the recombinant vector in the art can be used. In the present application, the gene can be obtained by synthesis of a biotechnology company. The separation and purification method is not particularly limited in the present application, and the conventional protein separation and purification method in the art can be used; the preferred technical solution is described in the examples.

[0037] The structure of collagen is characterized by using the circular dichroism (CD) commonly used in the art. CD is a spectroscopy method for determining the structure of a compound with chiral structure that can produce left and right optical difference absorption, and is mainly used for determining the asymmetry of molecular structure. Generally, biological macromolecules contain chiral groups and structures, so CD is often used to measure and observe the changes of structure and conformation of biological macromolecules. The CD characteristics of collagen triple helix structure are generally 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 the change of amino acid sequence and length. The thermal stability of protein is generally represented by the melting temperature (Tm), that is, the temperature at which 50% of the protein unfolds. For collagen, it refers to the temperature at which the triple helix structure is uncoiled and each forms a single chain, and the triple helix is uncoiled to 50%. Therefore, CD spectrum can be used to study the helical structure of collagen and the thermal denaturation process.

[0038] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[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 respectively, and were electroporated into Rosetta competent cells respectively, and were plated on solid LB plates (Cm + The final concentration was 34 μg / mL), and was incubated at 37℃ overnight. The wild type BaP4H single colony grown on the plate was picked into 4 mL liquid SOC medium (Chloramphenicol final concentration was 34 μg / mL), and was incubated at 37℃ overnight to extract plasmid. The pET28a-SEQ9 plasmid was extracted in the same way. pGRO-strep-BaP4H and pET28a-SEQ9 were co-transformed into Rosetta competent cells, and were plated on double-antibiotic LB solid medium (Chloramphenicol final concentration was 34 μg / mL, Kanamic final concentration was 50 μg / mL) and incubated overnight.

[0041] The single colony grown on the plate was picked into 10 mL double-antibiotic liquid LB medium (Chloramphenicol final concentration 34 μg / mL, Kanamic final concentration 50 μg / mL) and cultured overnight at 37°C. 10 mL of the bacterial solution was transferred into 100 mL double-antibiotic liquid LB medium and cultured overnight at 37°C. 10 mL of the overnight activated liquid bacterial strain was transferred into 1 L liquid LB medium at an inoculation amount of 1%, and was divided into 7 groups, and was induced for expression according to the culture conditions 1-7 in the table:

[0042] 7000rmp, 4°C centrifugation for 30 min to collect bacteria. Lysis Buffer was added to the bacteria at a ratio of bacteria amount: Lysis Buffer = 1:8 (W / V), and the bacteria were resuspended, and the cells were broken by a high-pressure homogenizer. The broken whole bacteria were 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 to 1 L of the bacterial solution, and the protein was purified by affinity chromatography, and after purification, the protein was dialyzed against potassium phosphate buffer. After dialysis, the protein was determined by CD.

[0043] The experimental results are shown in Figure 1. The best effect was obtained when 2 mg / mL of arabinose was added after 1 h of amplification, and the subsequent co-expression was carried out under this condition.

[0044] Example 2, co-expression of BaP4H mutant and recombinant collagen protein

[0045] 1. The mutant constructed in the application is a mutant of wild-type BaP4H, and the amino acid sequence of the wild-type BaP4H is as follows:

[0046] 2. The mutant constructed in the application and its number are shown in the following table:

[0047] 3. The nucleotide sequence and amino acid sequence involved in the application are as follows:

[0048] (1) When the mutation site is D50A+R73T:

[0049] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO. 2):

[0050] The amino acid sequence of the BaP4H mutant is (SEQ ID NO. 3):

[0051] (2) When the mutation site is D50A+D129K:

[0052] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO. 4):

[0053] The amino acid sequence of the BaP4H mutant is (SEQ ID NO. 5):

[0054] (3) When the mutation site is D50A+D152G:

[0055] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO. 6):

[0056] The amino acid sequence of the BaP4H mutant is (SEQ ID NO. 7):

[0057] (4) When the mutation site is D50A+H134T:

[0058] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO. 8):

[0059] The amino acid sequence of the BaP4H mutant is (SEQ ID NO. 9):

[0060] (5) When the mutation site is D50A+H114D:

[0061] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO. 10):

[0062] The amino acid sequence of the BaP4H mutant is (SEQ ID NO. 11):

[0063] (6) When the mutation site is D50A+R142E:

[0064] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO. 12):

[0065] The amino acid sequence of the BaP4H mutant is (SEQ ID NO. 13):

[0066] (7) When the mutation site is D50A+S81L:

[0067] The nucleotide sequence of the BaP4H mutant is (SEQ ID NO. 14):

[0068] The amino acid sequence of the BaP4H mutant is (SEQ ID NO. 15):

[0069] 4. The above mutants were constructed and extracted according to the method of Example 1, and co-transformed with pET28a-SEQ9 into Rosetta competent cells for protein expression and purification.

[0070] SDS-PAGE verified the expression of recombinant collagen (Figure 2), and Western Blot-strep verified the expression of BaP4H mutant (Figure 3).

[0071] Example 3, determination of the hydroxylation rate of recombinant collagen

[0072] The hydroxylation rate was determined using the Solybio Hydroxyproline (HYP) content detection kit.

[0073] The microplate reader was preheated for more than 30 min, and the wavelength was adjusted to 560 nm. The standard was diluted with ultrapure water to 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.234 μg / mL standard solution. After dialysis, the protein was uniformly diluted to 0.3 mg / mL, 6M HCl was added at 1:1 (V / V), mixed well, and sealed at 110°C for 8h. After cooling, adjust the pH to neutral with NaOH, and dilute to volume. The above samples were used to draw the standard curve and sample test according to the table:

[0074] Hydroxylation rate calculation: first calculate the proportion of proline in the protein sequence B (i.e. the proportion of proline in the middle amino acid), then calculate the protein concentration after dilution C (i.e. the protein is diluted by the extraction liquid and the neutralizing liquid, generally 3 times dilution); then the final formula after substituting x in the previous step is: hydroxylation rate (%) = x / (BxC) x 100%.

[0075] The experimental results show that the collagen co-expressed with the BaP4H mutant is hydroxylated, and the hydroxylation rates of C5 and C11 are 2.35 times and 1.66 times higher than those of the wild type, respectively.

[0076] Example 4, determination of the stability of hydroxylated recombinant collagen

[0077] The recombinant collagen stability test was determined by circular dichroism spectrometer. The protein samples after dialysis were diluted to the same concentration for standby. The dialysate was mixed with water at a ratio of 1:2 (V / V) as a blank group to determine the normal temperature CD baseline under the "Spectra Measurement" program at 190-260 nm; the protein was mixed with water at a ratio of 1:2 (V / V) as a test group. The dialysate was mixed with water at a ratio of 1:15 (V / V) as a blank group to determine the variable temperature CD baseline under the "Temperature Interval Measurement" program at 190-260 nm, 25-95℃, with a step of 1℃. The rest of the samples were determined in the same way. The normal temperature CD results showed that all the above mutants had a three-helix characteristic peak, 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 after hydroxylation by the wild-type BaP4H, preferably increased by 3℃, indicating that the efficient hydroxylation of the BaP4H mutant of the application to the recombinant collagen enhanced the stability of the collagen.

Claims

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

2. The BaP4H mutant as described in 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 as described in claim 2, characterized in that, The amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.3 when the mutation site is D50A+R73T; or, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.5 when the mutation site is D50A+D129K; or, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.7 when the mutation site is D50A+D152G; or, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.9 when the mutation site is D50A+H134T; or, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.11 when the mutation site is D50A+H114D; or, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.13 when the mutation site is D50A+R142E; or, the amino acid sequence of the BaP4H mutant is as shown in SEQ ID NO.15 when the mutation site is D50A+S81L.

4. The encoding gene of the BaP4H mutant according to claim 3, 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.

5. A recombinant expression vector, characterized in that, It contains the encoding gene as described in claim 4.

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

7. A recombinant cell, characterized in that, The recombinant cells contain the recombinant expression vector of claim 5, or have an exogenous encoding gene of claim 4 integrated into their chromosomes.

8. An enzyme preparation, characterized in that, It includes the BaP4H mutant as described in any one of claims 1 to 3.

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

10. The application as described in claim 9, characterized in that, The method involves co-expressing the BaP4H mutant with recombinant collagen to catalyze the hydroxylation of proline in the recombinant collagen, including the following steps: i) The encoding gene of the BaP4H mutant was constructed into the pGRO7-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. ii) Pick the single colonies that grow on the plate and culture them in liquid SOC double-antibiotic medium as seed culture; iii) After inoculating the seed culture into double-antibiotic LB liquid medium and culturing for 1 hour, add arabinose to a final concentration of 2 mg / mL and then add IPTG to a final concentration of 1 mM for induction culture.

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