WO2018107521A1 - T4 polynucleotide kinase recombinase and preparation method, expression gene, expression vector, and host cell of same - Google Patents

Abstract

Provided are a T4 polynucleotide kinase recombinase and a preparation method, an expression gene, an expression vector, and a host cell of the same. The T4 polynucleotide kinase recombinase comprises: (a) a polypeptide composed of the amino acid sequence shown by SEQ ID No. 1; (b) and a polypeptide having at least 98% homology with the polypeptide composed of the amino acid sequence shown by SEQ ID No. 1; or (c) a polypeptide obtained by deletion, replacement, or addition of one or more amino acids of the polypeptide composed of the amino acid sequence shown by SEQ ID No. 1. The T4 polynucleotide kinase recombinase is obtained by recombination using a genetic engineering technology.

Description

T4 polynucleotide kinase recombinase, preparation method thereof, expression gene, expression vector and host cell Technical field

The invention relates to the field of biotechnology, in particular to a T4 polynucleotide kinase recombinase, a preparation method thereof, an expression gene, an expression vector and a host cell.

Background technique

T4 Polynucleotide Kinase (T4 PNK), a polynucleotide 5' hydroxy kinase that catalyzes the gamma phosphate group of ATP to single or double stranded DNA, RNA, oligo 5' hydroxyl transfer of a nucleotide or a single nucleotide with a 3' phosphate group. Other NTPs can also produce the same reaction: 5'-OH+NTP→5'-P+NDP.

The above phosphorylation reaction is reversible. In the absence of ATP and the presence of ADP, T4 polynucleotide kinase can exhibit 5' phosphatase activity, catalyzing single- or double-stranded DNA, RNA, oligonucleotides or with 3' phosphate groups Transfer of the 5' phosphate group of a single nucleotide to ADP forms ATP. Other NTPs can also produce the same reaction: 5'-P+NDP→5'-OH+NTP (optimal pH is around 6.4).

When both ATP and ADP are present in moderation, T4 polynucleotide kinase can catalyze single- or double-stranded DNA, RNA, oligonucleotides or 5' phosphate groups of single nucleotides with a 3' phosphate group. Exchange reaction with the gamma phosphate group of ATP. Other NTPs can also produce the same reaction: 5'-P+NTP+NDP→5'-P+NDP+NTP.

T4 PNK also has 3' phosphatase activity, which can catalyze the dephosphorylation of 3' phosphorylated polynucleotide: 3'-P→3'-OH+Pi (optimum pH is about 5.9).

The kinase activity of T4 PNK is near the C-terminus, while the phosphatase activity is near the N-terminus.

T4 PNK is widely used, such as oligonucleotides, DNA or RNA 5' end labeling, as probes for Southern, Northern, EMSA, etc., gel electrophoresis markers, DNA sequencing primers, PCR primers, etc.; Phosphorylation of the 5' end of acid, DNA or RNA ensures that the subsequent ligation reaction proceeds smoothly; catalyzing the 5' phosphorylation of the 3' phosphorylated single nucleotide, allowing the single nucleotide to interact with the 3' end of the DNA or RNA Even The 3' terminal phosphate group is removed.

As a commonly used molecular biology reagent, the market demand for T4 PNK is huge. The recombinant T4 polynucleotide kinase cloned by genetic engineering technology is highly expressed in the pET system, but the following problems exist: easy expression and formation in the absence of soluble label In vivo, a small amount of active protein can be obtained by renaturation of inclusion bodies, but during storage, proteins tend to aggregate to form aggregates, forming precipitates with poor stability; increasing soluble label can increase the ratio of soluble protein, but the protein activity after purification is better. Low, if the label removal cost is high and the resulting active protein is less stable.

Summary of the invention

Based on this, it is necessary to provide a T4 polynucleotide kinase recombinase with better stability and higher activity.

It is also necessary to provide a method for producing the above T4 polynucleotide kinase recombinase, an expression gene, an expression vector, and a host cell.

A T4 polynucleotide kinase recombinase comprising:

(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID No. 1;

(b), a polypeptide having a polypeptide consisting of the amino acid sequence of SEQ ID No. 1 having at least 98% homology; or

(c) A polypeptide consisting of the amino acid sequence of SEQ ID No. 1, wherein one or more amino acids are deleted, replaced or increased.

An expression gene, including:

(a) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1, or a complementary strand of the polynucleotide;

(b) a polynucleotide having at least 98% homology with a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1, or a complementary strand of the polynucleotide;

(c) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1, wherein one or more amino acids are deleted, replaced or increased, and The polypeptide has beta-glucanase activity; or

(d) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide being SEQ ID The polypeptide consisting of the amino acid sequence shown in No. 1 has at least 98% homology.

An expression gene, including:

(a) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2, or a complementary strand of the polynucleotide;

(b), a polynucleotide having at least 98% homology to a polynucleotide comprising a nucleotide sequence represented by SEQ ID No. 2, or a complementary strand of said polynucleotide;

(c) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polynucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID No. 2, wherein one or more codons are deleted , substitute or increase.

An expression vector comprising the above expressed gene.

In one embodiment, the expression vector is pET-28a.

A host cell comprising the above gene expression vector, the host cell being a prokaryotic cell.

In one embodiment, the host cell is E. coli.

In one embodiment, the host cell is BL21.

A method for preparing a T4 polynucleotide kinase recombinase comprises the following steps:

Providing the above host cell, and inducing expression, collecting host cells after expression is completed;

The host cells after the expression is completed are lysed and centrifuged, and the supernatant is retained;

The supernatant was purified to obtain the T4 polynucleotide kinase recombinase.

In one embodiment, in the operation of inducing expression, the expression temperature is from 16 ° C to 20 ° C, the final concentration of the inducer is from 0.2 mM to 0.5 mM, and the time of induction of expression is from 12 h to 24 h.

The T4 polynucleotide kinase recombinase was recombined by genetic engineering technology. After expression, the T4 polynucleotide kinase recombinase has good stability and high activity.

DRAWINGS

Figure 1 is a SDS-PAGE (12%) analysis of recombinant T4 PNK protein in Example 2, wherein lane M is protein Marker, lanes 1 and 2 are before induction, and lanes 3 and 4 are induced whole-protein Expression, protein Marker molecular weight of 116kDa, 66kDa, 45kDa, 35kDa, 25kDa, 18.4kDa and 14.4kDa;

2 is a SDS-PAGE (12%) analysis diagram of recombinant T4 PNK in Example 2, wherein lane M is protein Marker, lane 1 is the original enzyme, lane 2 is diluted 5 times with the original enzyme, and lane 3 is diluted with the original enzyme. 10 times, Lane 4 is diluted 20 times with the original enzyme;

3 is a graph comparing the activity of recombinant T4 PNK and NEB-T4 PNK in Example 2 with SDS-PAGE (12%), wherein M is Marker, lane 1 is 100 U, lane 2 is 200 U, and lane 3 is 400 U. Lane 4 is 600U.

detailed description

The above described objects, features and advantages of the present invention will become more apparent from the detailed description. Numerous specific details are set forth in the description below in order to provide a thorough understanding of the invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention, and thus the invention is not limited by the specific embodiments disclosed below.

The invention discloses a T4 polynucleotide kinase recombinase, comprising:

(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID No. 1;

(b), a polypeptide having a polypeptide consisting of the amino acid sequence of SEQ ID No. 1 having at least 98% homology; or

(c) A polypeptide consisting of the amino acid sequence of SEQ ID No. 1, wherein one or more amino acids are deleted, replaced or increased.

The T4 polynucleotide kinase recombinase was recombined by genetic engineering technology. After expression, the T4 polynucleotide kinase recombinase has good stability and high activity.

The invention also discloses an expression gene of the above T4 polynucleotide kinase recombinase according to an embodiment, which comprises the followings:

(a) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1, or a complementary strand of the polynucleotide;

(b) a polynucleotide having at least 98% homology with a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1, or a complementary strand of the polynucleotide;

(c) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide being SEQ ID The amino acid sequence composition shown in No. 1, wherein one or more amino acids are deleted, substituted or increased, and the polypeptide has β-glucanase activity; or

(d) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide having at least 98% homology to a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1.

The invention also discloses another expression gene of the above T4 polynucleotide kinase recombinase, which comprises the following:

(a) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2, or a complementary strand of the polynucleotide;

(b), a polynucleotide having at least 98% homology to a polynucleotide comprising a nucleotide sequence represented by SEQ ID No. 2, or a complementary strand of said polynucleotide;

(c) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polynucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID No. 2, wherein one or more codons are deleted , substitute or increase.

The present invention also discloses an expression vector of the above T4 polynucleotide kinase recombinase according to an embodiment, which comprises the above-mentioned expression gene.

Preferably, the expression vector is pET-28a.

The present invention also discloses a host cell of the above T4 polynucleotide kinase recombinase according to an embodiment, comprising the above gene expression vector, wherein the host cell is a prokaryotic cell.

Preferably, the host cell is Escherichia coli.

More preferably, the host cell is E. coli BL21 (DE3) (purchased from Invitrogen, USA).

The invention also discloses a preparation method of the above T4 polynucleotide kinase recombinase according to an embodiment, comprising the following steps:

S10, providing the above host cell, and inducing expression, collecting the host cell after expression is completed.

The host cell can be obtained by genetic engineering technology, and the recombinant T4 PNK prokaryotic expression vector was constructed, and the codon-optimized T4 PNK gene was successfully cloned into the His-tagged pET vector to construct the above T4. The pET vector expressing the gene of the polynucleotide kinase recombinase is correctly transformed, and the pET vector containing the expression gene of the T4 polynucleotide kinase recombinase is transformed into a host cell and stored in glycerol.

In the operation of inducing expression, the expression temperature is 16 ° C to 20 ° C (preferably 18 ° C), the final concentration of the inducer is 0.2 mM to 0.5 mM (preferably 0.25 mM), and the time for inducing expression is 12 h to 24 h (preferably 16h).

After induction of expression, centrifugation, retention of the precipitate is the host cell after expression is completed.

S20. The host cells obtained after the expression of S10 are lysed and then centrifuged, and the supernatant is retained.

Specifically, the S20 is obtained by suspending and mixing the host cells after the expression is completed in an ice bath, and then lysing the cells by ice bath, centrifuging and retaining the supernatant.

The lysis buffer included 50 mM Tris, 1.5 M NaCl, 10 wt% glycerol, 0.1 wt% Triton X-100, 1 mM PMSF (phenylmethylsulfonyl fluoride), and 20 mM Imidazole (imidazole), and the pH of the lysis buffer was 7.5.

S30. Purifying the supernatant obtained in S20 to obtain a T4 polynucleotide kinase recombinase.

S30 is specifically: the supernatant is equilibrated by a lysis buffer, and the Ni column is fully equilibrated with the lysis buffer after the sample is completed, and then washed thoroughly with the pre-wash buffer; further eluting the protein with the elution buffer, the Ni affinity layer The analysis has removed a large portion of the heteroprotein, and ion exchange removes trace amounts of the heteroprotein and nucleic acid.

The prewash buffer included 50 mM Tris, 0.5 M NaCl, 10 wt% glycerol, 0.1 wt% Triton X-100, and 100 mM Imidazole, and the pH of the prewash buffer was 7.5.

The elution buffer included 50 mM Tris, 500 mM NaCl, 10 wt% glycerol, 0.1 wt% Triton X-100, and 0.5 M Imidazole, and the pH of the elution buffer was 7.5.

The gene of T4 PNK was cloned into the pET vector by vector construction, and the expression was induced under low temperature conditions. The soluble ratio of the expressed protein was increased by low temperature and low IPTG concentration, and the soluble ratio of the expressed protein was increased by using high salt lysis buffer. The solubility of the expressed protein is greatly increased. In addition, the purification of T4 PNK is simpler and more convenient due to the addition of His tag.

The T4 polynucleotide kinase recombinase was recombined by genetic engineering technology. After expression, the T4 polynucleotide kinase recombinase has good stability and high activity.

The following are specific examples.

The use of drugs and instruments in the examples, unless otherwise stated, is a routine choice in the art. Experimental methods in which no specific conditions are indicated in the examples are usually carried out according to conventional conditions, such as those described in the literature, in the book, or by the method recommended by the manufacturer of the kit.

Example 1. Expression and purification of recombinant T4 PNK

The recombinant T4 PNK gene was successfully constructed by recombinant T4 PNK prokaryotic expression vector, and the codon-optimized T4 PNK gene was successfully cloned into His-tagged pET vector to construct the expression gene containing T4 polynucleotide kinase recombinase. The pET vector (sequence as shown in SEQ ID No. 2) was transformed into the BL21 (DE3) (purchased from Invitrogen, USA) after the sequencing result was correct, and the pET vector containing the expression gene of the T4 polynucleotide kinase recombinase was transformed. In the case, the strain is obtained and stored in glycerin.

100 μL of the above strain was inoculated into 10 mL of LB medium containing 50 mg/mL kanamycin; cultured for 2-3 hours until the OD600 was 1.0 or so, and 5 mL of the bacterial solution was transferred to 2 bottles containing 50 mg/mL card. In 500 mL LB medium of nalpenicillin, the culture was continued for 3-4 hours until the OD600 was about 0.6. The expression of T4 PNK was induced by adding IPTG at a final concentration of 0.25 mM. The expression temperature was controlled at 18 ° C, and 16 cells were induced. After a few hours, the bacterial cells were collected. 1 L of the bacterial liquid was collected by centrifugation at 7000 rpm in a 400 mL centrifuge cup at 4 ° C for 3 min.

1 L of the bacterial cells were collected, suspended and mixed in a 100 mL ice bath lysis buffer containing 50 mM Tris, 1.5 M NaCl, 10% glycerol, 0.1% Triton X-100, 0.2 mM PMSF, pH 7.5, 20 mM Imidazole; ultrasonically lyse the cells in ice bath (retain 40 μL of the whole cell suspension), centrifuge to remove the supernatant (retain 40 μL of the bacterial cell disrupted supernatant), and resuspend the pellet with an equal volume of lysis buffer (retain 40 μL of bacteria) Body broken sediment). The supernatant was cleaved through a lysis buffer-balanced Ni column, and the flow-through solution was collected (retaining 40 μL of Ni-pass); after the completion of the sample, the solution was thoroughly equilibrated with a lysis buffer, and the flow-through solution was collected (retaining 40 μL of Ni-equilibrium); Wash well with pre-wash buffer containing 50 mM Tris, 0.5 M NaCl, 10% glycerol, 0.1% Triton X-100, pH 7.5, 100 mM Imidazole, and collect the flow through solution (retain 40 μL Ni-pre Wash); further elute the protein with elution buffer containing 50 mM Tris, 500 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.5 M Imidazole, pH 7.5, collect the flow through solution (retaining the sample) 40 μL of Ni- eluted) to obtain recombinant T4 PNK. Ni affinity chromatography has removed most of the heteroproteins, and ion exchange removes trace amounts of heteroproteins and nucleic acids.

Example 2, SDS-PAGE (12%) protein electrophoresis to verify the size and purity of T4 PNK protein

The recombinant T4 PNK obtained in Example 1 was subjected to SDS-PAGE protein electrophoresis detection to identify the size and purity of recombinant T4 PNK.

(1) Take 40 μL of each sample and add 5 μL of 5× Loading Buffer, boil water for 10 min, load 5 μL each with protein Marker, concentrate the gel at 100V, separate the gel at 180V, and test the Marsh bright blue to obtain Figure 1.

(2) Dilute the T4 PNK original enzyme solution by 5 times, 10 times and 20 times respectively, add 40 μL of the diluted enzyme solution and the original enzyme solution to 10 μL of 5× Loading Buffer, boil water for 10 min, load 10 μL, and concentrate the gel at constant pressure. 100V, separation gel constant pressure 180V, Coomassie brilliant blue staining, get Figure 2.

(3) The recombinant T4 PNK and NEB-T4 PNK obtained in Example 1 were separately diluted by the commercial T4 PNK (NEB-T4 PNK) of NEB, and each sample was 40 μL plus 5× Loading Buffer 10 μL. The boiling water bath was used for 10 min, and each sample was loaded with 5 μL of the protein Marker, and the concentrated gel was pressed at a constant pressure of 100 V, and the gel was separated at a constant pressure of 180 V, and the test was performed by Coomassie Brilliant Blue, and FIG. 3 was obtained.

The results of SDS-PAGE (12%) analysis showed that:

(1) Please refer to Figure 1, each electrophoresis strip is pressed into a straight line in the concentrated gel; from the strip position in Figure 1, combined with the sequencing results, the recombinant T4 PNK is correctly determined; 18 ° C, 0.25 mM IPTG induction, The expression level of recombinant T4 PNK can reach more than 10%, and the solubility can reach more than 80%.

(2) Ni affinity chromatography has removed most of the heteroproteins, and ion exchange can remove trace amounts of heteroproteins and nucleic acids.

(3) Please refer to Figure 2 to compare the intensity of the original band in the original enzyme and the main band after dilution. If the intensity of the miscellaneous band in the original enzyme < the intensity of the main band after dilution, the purity of the recombinant T4 PNK is greater than 95%, the background of the rubber surface is clean, the sample strip is clear, and the purity is qualified. As can be seen from Fig. 2, the purified recombinant T4 PNK has a molecular weight of about 35 kDa and a protein purity of more than 95%.

(4) Referring to Fig. 3, the recombinant T4 PNK obtained in Example 1 exhibited an enzyme activity similar to that of the commercial enzyme of NEB, and the specific activity was about 10 U/μg.

(5) A 1 L fermentation broth can obtain about 1 L of a 10 U/μL T4 PNK working solution.

Example 3: Determination of activity of recombinant T4 PNK enzyme

The activity of recombinant T4 PNK enzyme was determined by [γ- ³² P]ATP incorporation. The specific procedure reference: Richardson, CC (1971) Procedures in Nucleic Acids Res., 2, 815-826

Activity is defined as: Micrococcal Nuclease-treated calf thymus DNA as a substrate, required to incorporate 1 nmol of [γ- ³² P]ATP into an acid-insoluble precipitate at 37 ° C, pH 7.6 for 30 minutes. The amount of enzyme is defined as 1 unit of activity.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (10)

A T4 polynucleotide kinase recombinase characterized by comprising: (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID No. 1; (b), a polypeptide having a polypeptide consisting of the amino acid sequence of SEQ ID No. 1 having at least 98% homology; or (c) A polypeptide consisting of the amino acid sequence of SEQ ID No. 1, wherein one or more amino acids are deleted, replaced or increased. An expression gene characterized by comprising: (a) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1, or a complementary strand of the polynucleotide; (b) a polynucleotide having at least 98% homology with a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1, or a complementary strand of the polynucleotide; (c) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1, wherein one or more amino acids are deleted, replaced or increased, and The polypeptide has beta-glucanase activity; or (d) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide having at least 98% homology to a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1. An expression gene characterized by comprising: (a) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2, or a complementary strand of the polynucleotide; (b), a polynucleotide having at least 98% homology to a polynucleotide comprising a nucleotide sequence represented by SEQ ID No. 2, or a complementary strand of said polynucleotide; (c) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polynucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID No. 2, wherein one or more codons are deleted , substitute or increase. An expression vector comprising the expression gene of claim 2 or 3. The gene expression vector according to claim 4, wherein the expression vector is pET-28a. A host cell comprising the gene expression vector of claim 4 or 5, wherein the host cell is a prokaryotic cell. The host cell according to claim 6, wherein the host cell is Escherichia coli. The host cell according to claim 7, wherein the host cell is BL21. A method for preparing a T4 polynucleotide kinase recombinase, comprising the steps of: Providing the host cell according to any one of claims 6 to 8, and inducing expression, collecting host cells after expression is completed; The host cells after the expression is completed are lysed and centrifuged, and the supernatant is retained; The supernatant was purified to obtain the T4 polynucleotide kinase recombinase. The method for producing a T4 polynucleotide kinase recombinase according to claim 9, wherein in the operation of inducing expression, the expression temperature is 16 ° C to 20 ° C, and the final concentration of the inducer is 0.2 mM. At 0.5 mM, the time to induce expression was 12 h to 24 h.

Images