CN108588047B

CN108588047B - Homocysteine methyltransferase mutant and application thereof, nucleic acid, expression vector, host cell and reagent

Info

Publication number: CN108588047B
Application number: CN201810321831.7A
Authority: CN
Inventors: 彭毅; 王晓霞
Original assignee: Suzhou Huizhen Biotechnology Co ltd
Current assignee: Zhejiang Weijing Biotechnology Co.,Ltd.
Priority date: 2018-04-11
Filing date: 2018-04-11
Publication date: 2021-09-03
Anticipated expiration: 2038-04-11
Also published as: CN108588047A

Abstract

The invention adopts error-prone PCR and site-directed mutagenesis to screen out a homocysteine methyltransferase mutant, which contains one or more than two amino acid mutations at amino acids D53, D283, L252 and Q219 on the basis of wild HMT. The mutant has doubled activity and improved stability. The invention also discloses a nucleic acid for coding the homocysteine methyltransferase mutant, an expression vector comprising the nucleic acid, a host cell comprising the expression vector, and a reagent comprising at least one of the homocysteine-S-methyltransferase mutant, the nucleic acid, the expression vector and the host cell.

Description

Homocysteine methyltransferase mutant and application thereof, nucleic acid, expression vector, host cell and reagent

Technical Field

The invention relates to an enzyme and application thereof, nucleic acid for coding the enzyme, an expression vector comprising the nucleic acid, a host cell comprising the expression vector, and a reagent comprising at least one of the enzyme, the nucleic acid, the expression vector and the host cell. More specifically, the invention relates to a homocysteine methyltransferase mutant and application thereof, nucleic acid for coding the homocysteine methyltransferase mutant, an expression vector comprising the nucleic acid, a host cell comprising the expression vector, and a reagent comprising at least one of the homocysteine methyltransferase mutant, the nucleic acid, the expression vector and the host cell.

Background

Methyltransferases catalyze the transfer of the methyl group on S-adenosylmethionine (SAM) to substrate molecules to produce a variety of important physiologically active substances containing methyl groups, achieving the purpose of function regulation.

Homocysteine methyltransferase (HMT, EC 2.1.1.10) is one of methyltransferases, can catalyze homocysteine and SAM to react to generate S-adenosylhomocysteine and methionine, can be used for preparing Homocysteine (HCY) biochemical diagnostic reagents, and is a key raw material enzyme.

The existing HMT is an amino acid sequence of Saccharomyces cerevisiae SAM4, and has low activity and poor stability.

In view of the above problems, chinese patent CN102391999B discloses a homocysteine methyltransferase, a nucleotide sequence thereof, a recombinant vector, a recombinant host cell, a preparation method and a kit, which is obtained from Candida albicans (Candida albicans sc5314) and has the sequence shown in SEQ ID NO: 1, and obtaining a homocysteine methyltransferase with an amino acid sequence shown in SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof. The two homocysteine methyltransferases obtained have good heat resistance and can keep higher stability in a wide temperature range. Both homocysteine methyltransferases have a relative activity of more than 60% after 15 minutes of standing in a buffer at pH6.0 at 40 ℃.

However, the homocysteine methyltransferase in the scheme only improves the heat resistance, but the enzyme is usually required to be placed at 4 ℃ for several months or even calculated according to years, the stability at the high temperature of 40 ℃ is necessarily good, and in the scheme, only 60 percent of relative activity is relatively low after 15min at 40 ℃, and the overall activity is relatively low.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a homocysteine methyltransferase mutant which obviously improves the heat resistance, activity and stability of enzyme.

The technical purpose of the invention is realized by the following technical scheme: a mutant homocysteine methyltransferase, which contains one or more amino acid mutations at amino acids D53, D283, L252 and Q219 on the basis of wild-type HMT, wherein the amino acid mutation at D53, D283, L252 and Q219 is any one of 19 amino acids excluding the original amino acid.

By adopting the technical scheme, the directional mutation is carried out on a specific amino acid position through theoretical knowledge and experience, and compared with a Wild Type (WT), the activity and the stability of the mutant are improved to a certain extent, and the heat resistance is also improved.

The invention is further configured to: the mutant homocysteine methyltransferase is mutated into glutamic acid, asparagine or glycine at D53 and D283 amino acids.

The invention is further configured to: the homocysteine methyltransferase mutant is mutated into glutamic acid at D53 amino acid, and is mutated into asparagine at D283 amino acid.

The invention is further configured to: the homocysteine methyltransferase mutant is mutated into isoleucine, valine, phenylalanine or lysine at an L252 amino acid; the homocysteine methyltransferase mutant is mutated at amino acid Q219 to glutamate, aspartate or asparagine.

The invention is further configured to: homocysteine methyltransferase mutants contain amino acid mutations at other amino acid positions.

The invention also aims to provide a nucleic acid, and the activity and stability of the homocysteine methyltransferase mutant obtained by coding are obviously improved compared with those of wild HMT.

The second technical purpose of the invention is realized by the following technical scheme: a nucleic acid encoding a mutant homocysteine methyltransferase, said nucleic acid encoding a mutant homocysteine methyltransferase described in the above schemes.

The third purpose of the invention is to provide an expression vector, and the activity and stability of the homocysteine methyltransferase mutant contained in the expression vector are obviously improved compared with those of wild HMT.

The third technical purpose of the invention is realized by the following technical scheme: an expression vector comprising the nucleic acid of the above scheme.

The invention also aims to provide a host cell, wherein the activity and stability of the homocysteine methyltransferase mutant contained in the host cell are obviously improved compared with those of wild HMT.

The fourth technical purpose of the invention is realized by the following technical scheme: a host cell comprising the expression vector of the above protocol.

The invention is further configured to: the host cell is a prokaryotic cell.

The invention is further configured to: the host cell is an escherichia coli cell.

The invention is further configured to: the host cell is Escherichia coli BL21 (DE 3) cell.

The fifth purpose of the invention is to provide the application of the homocysteine methyltransferase mutant, which catalyzes the reaction of S-adenosylmethionine and homocysteine to generate S-adenosylhomocysteine and methionine with higher efficiency.

The fifth technical purpose of the invention is realized by the following technical scheme: in the scheme, the homocysteine-S-methyltransferase mutant is used for catalyzing the reaction of S-adenosylmethionine and homocysteine to generate S-adenosylhomocysteine and methionine.

A homocysteine detection reagent, which contains the homocysteine methyltransferase mutant in the scheme.

By adopting the technical scheme, the using amount of HMT can be reduced, the service life of the Hcy reagent can be prolonged, the production cost is reduced, the calibration times are reduced, and the effective detection number of the reagent is increased.

In conclusion, the invention has the following beneficial effects:

firstly, the invention optimizes and synthesizes the nucleic acid sequence for coding HMT according to the codon preference of escherichia coli, compared with the sequence from a wild strain, the optimized gene sequence is more favorable for expression in the escherichia coli, the expression quantity is high, the recombinant plasmid is stable, the repeatability is good, and the loss is not easy.

Secondly, through multiple rounds of tests, a large number of combinations are carried out, the enzyme activity and the stability are detected, and the results show that when 3 positions, namely the D283 amino acid mutation is asparagine, the L252 amino acid mutation is isoleucine and the Q219 amino acid mutation is aspartic acid, are mutated simultaneously, the mutants with obviously improved enzyme activity and stability are obtained, finally, the enzyme activity is doubled, the stability is also greatly improved, and the enzyme activity is improved from 75 percent of a wild type to about 90 percent.

Thirdly, the invention selects pET-28a (+) expression vector, selects BL21 (DE 3) cell for expression, pET series vector with T7 promoter, T7 expression system can use strong bacteriophage T7 promoter for high level expression. It is very suitable for the expression of soluble, non-toxic recombinant proteins in E.coli. The pET-28a-HMT obtained by the invention obtains obvious high-level expression when being expressed. The expression of the T7 RNA polymerase gene is controlled by lacUV5 promoter of the DE3 region of lambda phage, and is very suitable for expression systems of T7 and T7Lac promoters, such as pET, pEASY and the like. The expression vector of pET-28a series is preferably selected in the invention, and the paired BL21 (DE 3) expression cells are very beneficial to the expression of HMT protein with high soluble expression quantity. The optimized combination of the carrier and the cells obtains soluble target protein with high expression level through experiments.

Thirdly, the invention directly adopts the principle of the Hcy detection kit, and the activity of HMT is detected in turn after combination, so that the influence of the activity change of the enzyme in the Hcy kit on the kit is more direct, and the problem of inconsistency of simple enzyme activity detection and enzyme activity detection in the kit can be avoided.

Finally, a series of experiments show that the service life of the Hcy reagent can be prolonged while the usage amount of HMT is reduced, so that the production cost is reduced, the calibration times are reduced, and the effective detection amount of the reagent is increased.

Detailed Description

The SEQ ID NO.1 of the invention is an HMT amino acid sequence from saccharomyces cerevisiae, the SEQ ID NO.2 is an optimized HMT nucleotide sequence of the invention, and the sequence table is shown in Table 5. SEQ ID NO.3-17 are the HMT mutant amino acid sequences of the present invention.

The invention obtains a series of HMT mutants by optimizing an amino acid sequence of HMT (human papillomavirus) from saccharomyces cerevisiae as shown in SEQ ID NO.1 through an escherichia coli codon and a nucleotide sequence as shown in SEQ ID NO.2 through random mutation and site-specific mutation, wherein the HMT mutants as shown in SEQ ID NO.3-15 have better enzyme activity, and meanwhile, the HMT mutants as shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.14 and SEQ ID NO.15 have obviously better stability.

The invention provides an HMT mutant, wherein the amino acid sequence of the HMT mutant is,

the mutation points in the SEQ ID NO.3 sequence are as follows: D53E

The mutation points in the SEQ ID NO.4 sequence are: D283N

The mutation points in the SEQ ID NO.5 sequence are: L252I

The mutation points in the SEQ ID NO.6 sequence are: Q219D

The mutation points in the SEQ ID NO.7 sequence are: D53E + L252I

The mutation points in the SEQ ID NO.8 sequence are: D53E + D283N

The mutation points in the SEQ ID NO.9 sequence are: D53E + Q219D

The mutation points in the SEQ ID NO.10 sequence are: D283N + L252I

The mutation points in the SEQ ID NO.11 sequence are: D283N + Q219D

The mutation points in the SEQ ID NO.12 sequence are: L252I + Q219D

The mutation points in the SEQ ID NO.13 sequence are: D53E + L252I + D283N

The mutation points in the SEQ ID NO.14 sequence are: D53E + L252I + Q219D

The mutation points in the SEQ ID NO.15 sequence are: D283N + L252I + Q219D

The gene containing the mutant was ligated into pET-28a (+), and expressed in the host cell Escherichia coli BL21 (DE 3) to obtain the mutant protein.

The present invention is further illustrated below by reference to the tables and examples, except that the reagents and media used in the present invention are commercially available.

Example 1 construction of HMT mutation library by error-prone PCR (error-prone PCR)

By usingGeneThe Morph II random mutagenesis kit amplifies an HMT gene by taking an optimized HMT (shown as SEQ ID NO. 2) nucleotide sequence as a template, and randomly introduces mutations.

Amplification primers

F：5’-CATGCCATGGCTAGATTGCCATTGAAG (NcoI endonuclease underlined)

R：5’-CCGCTCGAGAGTGTACTTCTTAACAGCAG (XhoI endonuclease underlined)

Reaction conditions are as follows: the reaction conditions are as follows: pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 60s and extension at 72 ℃ for 2min, 25 cycles in total, performing 0.8% agarose electrophoresis, and recovering the target gene fragment by using the kit. The ligation reaction was carried out using NcoI and XhoI double-digested according to the method described in the product manual of NEB, and then with pET-28a (+) vector (kana resistance) digested with NcoI and XhoI under the following conditions: the vector and fragments were mixed in a molar ratio of 1:3, and 0.5. mu.L of Takara T4 DNA ligase was added overnight at 16 ℃. Transformation into by electric shockE.coliHST08 Premium Electro Cells, obtained over 10⁵Pools of mutants from individual clones.

Example 2 screening of a library of HMT mutants

The mutant pool clones of example 1 were collected, extracted to obtain plasmids, transferred to E.coli expression strain BL21 (DE 3), spread on a Kana-containing LB plate, and cultured for 12 hours. Single clones were picked in 96-well plates containing 150. mu.L of TB medium (containing 50. mu.g/mL kanamycin, 1mM IPTG), 37 ℃ at 245rpm, and shake-cultured for 36 h. Each monoclonal was replicated in a 96-well plate replicator on LB solid medium plate, and after 12 hours of culture at 37 ℃, it was stored in a 4 ℃ refrigerator. The cell culture in each well of the 96-well plate was gently aspirated by a line gun and dispensed in the corresponding position into the 96-well plates of the A-plate and the B-plate, and the culture in each well of each 96-well plate was 70. mu.L. Centrifuging at 4 deg.C for 10min at 4000rpm, discarding supernatant, repeatedly freezing and thawing the bacterial cells in each well for 5 times, and resuspending with 30 μ L of 50mM pH7.6 sodium phosphate buffer. Directly adding Hcy detection reagents R1124 mu L and R232 mu L without HMT into the A plate, adding an enzyme labeling instrument, uniformly mixing, detecting the reaction condition, and screening out the mutant with the enzyme activity higher than that of a wild type control.

Example 3 stability testing

Mutants in plate B corresponding to increased activity in plate A were tested for stability.

Screening 4 mutants with influence on activity, sequencing the mutants by single clone, and obtaining the amino acid sequence shown in SEQ ID NO. 3-SEQ ID NO.6

The four mutation positions are D53, D283, L252 and Q219 respectively.

Example 4 site-directed random mutagenesis

According to the error-prone PCR screening result, selecting D53, D283, L252 and Q219 which have influence on the activity as mutation positions, adopting QuikChange XL Site-Directed Mutagenesis Kit, amplifying an HMT gene by taking the optimized HMT (see SEQ ID NO. 2) nucleotide sequence as a template, and introducing random mutation.

Amplification primers

D53-F：

5'-CAATCTTTCTGTTAGCAGAAGACTCNNNAGACCAGAAAGACTCAGAGATGAAT-3'

D53-R：

5'-ATTCATCTCTGAGTCTTTCTGGTCTNNNGAGTCTTCTGCTAACAGAAAGATTG-3'

D283-F：

5'-CAACAGTGTCCCAAGAGTTCAACTTNNNAGAGTTTGGCAACCAAATCTTCTTC-3'

D283-R：

5'-GAAGAAGATTTGGTTGCCAAACTCTNNNAAGTTGAACTCTTGGGACACTGTTG-3'

L252-F：5'-CATGTTTGGCAAAGCTTGGTGNNNAGACTCCAAGATGTCTGGAGA-3'

L252-R：5'-TCTCCAGACATCTTGGAGTCTNNNCACCAAGCTTTGCCAAACATG-3'

Q219-F：5'-TTGTCACCCAAGTCCTTGATAACNNNAGCAATCTCCTCCATAGTAGTAC-3'

Q219-R：5'-GTACTACTATGGAGGAGATTGCTNNNGTTATCAAGGACTTGGGTGACAA-3'

And (3) PCR system: 10 XBuffer 5. mu.L, pET28-HMT template 2. mu.L, primers (10. mu.M) 2. mu.L each, dNTP (2.5 mM) 2. mu.L, Quiksolution 3. mu.L, pfu (2.5U/. mu.L) 1. mu.L, and ultrapure water to make up 50. mu.L.

Amplification conditions: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 50s, annealing at 60 ℃ for 50s, and extension at 68 ℃ for 7min for 18 cycles, and extension at 68 ℃ for 7 min. The PCR product was treated with 1. mu.L of Dpn I and treated at 37 ℃ for 1 h. 10 μ L of the strain was transferred to E.coli DH5a by chemical method. The extracted plasmid was transferred into expression strain E. coli BL21 (DE 3). Enzyme activity was measured according to example two, and stability was measured according to example three. The specific data are shown in Table 1.

Table 1 the enzyme activity and stability of the positive mutants screened after the first round of site-directed random mutagenesis, WT is wild type.

Note:

1. mutant representation methods: wild-type amino acid-mutated position-mutated amino acid;

2. "-" merely indicates that the reaction is a reaction in which the absorbance is downward, and the absolute value of the numerical value indicates the level of the enzyme activity;

3. the reactivity of the samples each accelerated at 37 ℃ for 7 days was compared with the reactivity of the samples stored at 4 ℃ and the percentage indicates the percentage of the activity of the enzyme retained after acceleration.

In the series of technical schemes, through theoretical knowledge and experience, directional mutation is carried out on a specific amino acid position, in the first round of random mutation screening, mutants with obviously changed enzyme activity are firstly screened, compared with a Wild Type (WT), the enzyme activity is improved, some mutants basically keep unchanged, some mutants reduce and the stability is changed variously, and finally, the mutants with the enzyme activity improved by 50% or more are selected as a basis for further downward tests, namely, the D53 amino acid is mutated into glutamic acid, the D283 amino acid is mutated into asparagine, the L252 amino acid is mutated into isoleucine, and the Q219 amino acid is mutated into aspartic acid.

Example fifth second round of site-directed random mutagenesis

And (3) taking the positive clone with the improved activity and stability of the first round of site-specific random mutation as a template, and optionally taking one of the other three mutation positions as a primer to carry out the second round of site-specific random mutation. Other reference is made to example four.

The combination is as follows:

the D53 and the L252 are,

the positions of D53 and D283 are,

the sum of the values of D53 and Q219,

the sum of the values of D283 and L252,

the sum of the values of D283 and Q219,

l252 and Q219.

The enzyme activity and stability after the second round of site-directed random mutagenesis are shown in Table 2.

Table 2 the enzyme activity and stability of the positive mutants screened after the second round of site-directed random mutagenesis, WT is wild type.

Note:

Also, we selected mutants with improved enzyme activity of 77% or more and better stability as basis for further experiments.

EXAMPLE sixth round of site-directed random mutagenesis

And (3) taking the positive clone with the improved activity and stability of the second round of site-specific random mutation as a template, and optionally taking one of the other two mutation positions as a primer to carry out the second round of site-specific random mutation. Other reference is made to example four.

The combination is as follows:

d53, L252 and D283,

d53, L252 and Q219,

d53, D283 and Q219,

l252, D283 and Q219.

The enzyme activity and stability after the third round of site-directed random mutagenesis are shown in Table 3.

Table 3 shows the enzyme activity and stability of the positive mutants screened after the third round of site-directed random mutagenesis, WT is a wild type.

Note:

Also, the mutants with the enzyme activity improved by 90% or more and better stability are selected as a basis for further experiments.

Example seventh fourth round of fixed-point random mutagenesis

And taking the positive clone with the improved activity and stability of the third round of site-directed random mutagenesis as a template, and taking the primer of the position without the site-directed random mutagenesis as a primer to carry out the second round of site-directed random mutagenesis. Other reference is made to example four.

The combination is as follows: d53, D283, L252, Q219.

The enzyme activity and stability after the fourth round of site-directed random mutagenesis are shown in Table 4.

TABLE 4 enzyme activity and stability of the positive mutants screened after the fourth round of site-directed random mutagenesis, WT was wild type.

Note:

Through the experimental analysis, the 13 mutants have better enzyme activities compared with wild HMT, and the activities of the corresponding mutants of SEQ ID NO.3-15 are respectively improved by 1.49 times, 1.75 times, 1.80 times, 1.71 times, 1.89 times, 1.83 times, 1.78 times, 1.96 times, 1.92 times, 1.94 times, 2.00 times, 1.90 times and 2.20 times; wherein, the stability of SEQ ID NO.5, SEQ ID number 6, SEQ ID number 9, SEQ ID number 12, SEQ ID number 14 and SEQ ID NO.15 is outstanding, the stability is respectively improved to 85%, 81%, 85%, 80% and 90% compared with that of the wild type (75%), and the advantages of reducing the production cost, prolonging the shelf life and increasing the effective detection number in the Hcy detection reagent are shown.

In conclusion, through a large number of combinations, the enzyme activity and the stability are detected, and the results show that when 3 positions, namely asparagine at the D283 amino acid position, isoleucine at the L252 amino acid position and aspartic acid at the Q219 amino acid position, are mutated simultaneously, the mutant with obviously improved enzyme activity and stability is obtained, finally, the enzyme activity is doubled, the stability is also greatly improved, and the enzyme activity is improved to about 90 percent from 75 percent of a wild type.

The stability test of the enzyme at high temperature is generally carried out at 42 ℃ and 37 ℃ in the industry, or is carried out at 40 ℃, the specific temperature is not standard, an accelerated experiment of placing the enzyme at 37 ℃ for 7 days is adopted to verify the stability of the enzyme (see each mutant enzyme activity and stability data table), the mutation with good effect is mentioned in the invention, the relative activity of the protein placed at 37 ℃ for 7 days and the protein placed at 4 ℃ can be kept more than 80%, and the effect is quite excellent.

An expression vector comprising the nucleic acid of the above examples, preferably a pET series vector of the present invention, with a T7 promoter, can be used for high level expression in the T7 expression system using the strong bacteriophage T7 promoter. It is very suitable for the expression of soluble, non-toxic recombinant proteins in E.coli. The pET-28a-HMT obtained by the invention obtains obvious high-level expression when in expression, and the average yield of protein in thalli after per gram centrifugation is purified to obtain at least about 30-40mg of target protein with the purity of at least 93 percent.

A host cell, the activity and stability of homocysteine-S-methyltransferase mutant contained in the host cell are obviously improved compared with wild HMT; the host cell is a prokaryotic cell, preferably an Escherichia coli cell, and more preferably an Escherichia coli BL21 (DE 3) cell. The invention preferably selects BL21 (DE 3) cells, which are characterized in that the expression of the T7 RNA polymerase gene is controlled by lacUV5 promoter of the DE3 region of lambda phage, and the promoter is very suitable for expression systems of T7 and T7Lac promoters, such as pET, pEASY and the like. The expression vector of pET-28a series, paired with BL21 (DE 3) expression cell, is very favorable for the expression of HMT protein with high soluble expression quantity.

For expression vectors other than T7 promoter (such as pGEX of Tac promoter or pMAL series expression vectors), BL21 expression strain can be selected.

The invention relates to a homocysteine detection reagent, which contains homocysteine-S-methyltransferase mutant described in the above embodiment, the invention directly adopts the principle of Hcy detection kit, and the activity of HMT is detected after combination, so that the influence of activity change of the enzyme in the Hcy kit on the kit is more direct, thus avoiding the problems of simple enzyme activity detection and activity detection of the enzyme in the kit, and simply speaking, the kit components are complex, and the simple enzyme activity detection indicates that the enzyme activity is high, and the activity of the whole kit can be certainly improved when the enzyme activity can not be directly indicated to be actually applied in the kit.

TABLE 5 sequence Listing of SEQ ID NO.1 to SEQ ID NO.6

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims

1. A mutant homocysteine methyltransferase which is characterized by: the compound contains one or more amino acid mutations at amino acids D53, D283, L252 and Q219 on the basis of wild-type HMT, and the amino acids mutated at D53, D283, L252 and Q219 are respectively glutamic acid, asparagine, isoleucine and aspartic acid; the wild type HMT is an amino acid sequence shown in SEQ ID NO.1 of HMT derived from saccharomyces cerevisiae, and is optimized by an escherichia coli codon to be a nucleotide sequence shown in SEQ ID NO. 2.

2. A nucleic acid encoding a mutant homocysteine methyltransferase according to claim 1 wherein said nucleic acid is a nucleic acid encoding a mutant homocysteine methyltransferase.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. The host cell of claim 4, wherein the host cell is an E.coli cell.

6. The host cell of claim 4, wherein the host cell is an E.coli BL21 (DE 3) cell.

7. Use of the mutant homocysteine methyltransferase of claim 1 to catalyze the reaction of S-adenosylmethionine with homocysteine to produce S-adenosylhomocysteine and methionine.

8. A homocysteine detection reagent comprising a mutant homocysteine methyltransferase according to claim 1.