T4 DNA ligase mutants with improved ligation fidelity

Abstract

The invention belongs to the technical field of biology, and particularly relates to a T4 DNA ligase mutant with improved connection fidelity. The T4 DNA ligase mutant is obtained by mutating any one or more amino acid sites of lysine at the 365th site and lysine at the 367th site of wild type T4 DNA ligase. Compared with a wild type T4 DNA ligase, the T4 DNA ligase has higher fidelity, and the fidelity of the T4 DNA ligase is maximally improved to more than 10 times of that of the wild type T4 DNA ligase.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to a T4 DNA ligase mutant with improved ligation fidelity. BACKGROUND

[0002] T4 DNA ligase is an enzyme commonly used in DNA ligation reactions, widely used in gene cloning, recombinant DNA technology, nucleic acid detection and other applications. This is because T4 DNA ligase not only has the characteristics of high activity and good stability, but also can recognize various substrate types, including nicked double-stranded DNA (nDNA), cohesive end DNA and blunt end DNA.

[0003] However, the enzyme has high tolerance to mismatches, which easily leads to non-specific ligation, affecting the accuracy of experimental results, especially in applications requiring high precision ligation. Although a variety of high-fidelity ligases have been discovered and identified to improve the accuracy of nucleic acid ligation, they have very low activity in the ligation of cohesive end DNA substrates, which cannot be effectively applied, such as genome assembly. Therefore, improving the ligation fidelity of T4 DNA ligase and enhancing its accuracy in DNA ligation reactions is of great significance for scientific research and biotechnology applications. SUMMARY

[0004] The purpose of the present application is to provide a T4 DNA ligase mutant with improved ligation fidelity.

[0005] The technical solution adopted by the present application is:

[0006] The mutant of T4 DNA ligase or its polypeptide fragment with DNA ligation activity has one or more amino acid mutations at the following positions based on the wild-type T4 DNA ligase, the mutation positions are lysine at position 365 and / or lysine at position 367, and the entire amino acid sequence identification number is: K365A (SEQ ID NO: 2), K367A (SEQ ID NO: 3). These mutations are identified as having enhanced ligation fidelity.

[0007] The present application also includes T4 DNA ligase mutants, which have one or more amino acid substitutions and / or deletions and / or additions in addition to the above-mentioned amino acid mutations, and have substantially the same ligase activity and performance.

[0008] The present application further includes the DNA sequence before each amino acid sequence of the above-mentioned mutant (i.e. SEQ ID NO: 5 and SEQ ID NO: 6).

[0009] The present application further comprises degenerate nucleic acid sequences encoding each of (i) the above-mentioned T4 DNA ligase mutants, (ii) each of the proteins or polypeptide fragments having substantially the same enzymatic activity and performance as the above-mentioned T4 DNA ligase mutants with substitution and / or deletion and / or addition of one or more amino acids outside the above-mentioned T4 DNA ligase mutants.

[0010] The present application further comprises expression vectors of the above-mentioned T4 DNA ligase mutants.

[0011] The present application further comprises host bacteria of the above-mentioned T4 DNA ligase mutants.

[0012] The present application further comprises expression or protein construction methods of the above-mentioned T4 DNA ligase mutants.

[0013] The present application further comprises applications of the above-mentioned T4 DNA ligase mutants in DNA-DNA, DNA-RNA, RNA-RNA ligation. BRIEF DESCRIPTION OF DRAWINGS

[0014] FIG. 1A 、 FIG. 1B and FIG. 1C show the results of fidelity test gel electrophoresis (left panel) and its quantification (right panel) of wild type, K365A mutant, K367A mutant T4 DNA ligase, respectively. From left to right in each gel are the samples of reaction products of reactions with increasing reaction time. FIG. 1D The fidelity of ligation of wild type, K365A mutant, K367A mutant T4 DNA ligase is shown. The size of the fidelity of enzyme ligation is defined as the ratio of the initial velocity of the correct ligation to the mispair ligation. The quantification of the images shows the average and standard deviation of three parallel experiments. DETAILED DESCRIPTION

[0015] The specific embodiments of the present application will be further described with reference to the drawings, but the description of the embodiments is not intended to limit the scope of the present application in any way.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0017] The materials and instruments used in the following examples can be obtained from conventional commercial channels, unless otherwise specified.

[0018] Example 1: Construction and obtaining of mutant T4 DNA ligase

[0019] Wild type T4 DNA ligase, whose amino acid sequence is shown as SEQ ID NO. 1, has poor selectivity for ligation substrates and low sensitivity to mispaired substrates. Therefore, we carried out protein modification on wild type T4 DNA ligase and screened out T4 DNA ligase mutants with high fidelity.

[0020] The gene of T4 DNA ligase (bacteriophage T4) was codon-optimized to obtain its encoding nucleic acid sequence (SEQ ID NO: 4) for expression in E. coli, synthesized and cloned into pET28a expression vector. The vector encoding wild type T4 DNA ligase was subjected to site-directed mutagenesis using a site-directed mutagenesis kit (Jinsabio) to obtain expression vectors encoding K365A and K367A mutants. The plasmids were transformed into E. coli BL21 (DE3) competent cells for expression of wild type and mutant T4 DNA ligases. Cells were grown in Luria-Bertani (LB) medium containing 50 μg·mL-1kanamycin at 37 °C for 4 h, followed by addition of 0.2 mM isopropyl-D-thiogalactopyranoside (IPTG) and incubation at 18 °C for 20 h. Cells were collected by centrifugation and disrupted by a high-pressure homogenizer (Xinzhi Bio) in buffer A (50 mM HEPES, pH 8.0; 500 mM NaCl; 30 mM imidazole). After high-speed centrifugation, the supernatant was loaded onto a nickel column (Histrap FF, GE Healthcare) and washed with buffer containing 60 mM imidazole. Then, the bound protein was eluted with buffer B (50 mM HEPES, pH 8.0; 500 mM NaCl; 300 mM imidazole). After desalting using an ultrafilter, the protein was quantified using a Bradford protein kit (Bi Yun Tian Bio) and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the protein solution was mixed with an equal volume of glycerol and stored at -20 °C. -1 Cells were grown in Luria-Bertani (LB) medium containing 50 μg·mL-1kanamycin at 37 °C for 4 h, followed by addition of 0.2 mM isopropyl-D-thiogalactopyranoside (IPTG) and incubation at 18 °C for 20 h. Cells were collected by centrifugation and disrupted by a high-pressure homogenizer (Xinzhi Bio) in buffer A (50 mM HEPES, pH 8.0; 500 mM NaCl; 30 mM imidazole). After high-speed centrifugation, the supernatant was loaded onto a nickel column (Histrap FF, GE Healthcare) and washed with buffer containing 60 mM imidazole. Then, the bound protein was eluted with buffer B (50 mM HEPES, pH 8.0; 500 mM NaCl; 300 mM imidazole). After desalting using an ultrafilter, the protein was quantified using a Bradford protein kit (Bi Yun Tian Bio) and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the protein solution was mixed with an equal volume of glycerol and stored at -20 °C.

[0021] Example 2: Ligation fidelity assay

[0022] The standard ligation assay mixture consists of lx ligase buffer (40 mM Tris-HCl, pH 8.0; 10 mM MgCl2; 10 mM DTT; 0.2 mM ATP), T4 DNA ligase, dsDNA substrate. The reaction was performed at 37 °C for different time gradient, terminated with equal volume of electrophoresis loading buffer (40 mM EDTA, 8 M urea, 30% glycerol and 0.01% xylene cyanol FF), and the samples were separated by urea denaturing polyacrylamide gel electrophoresis (Urea-PAGE). The electrophoresis bands were quantified by gray value, and the yield of each time point was calculated by the gray value of substrate and product, and the initial velocity of ligation was calculated according to the yield of different time points. According to the initial velocity of ligation yield of the correct and mismatched ligation, the ligation fidelity was calculated.

[0023] FIG. 1A , FIG. 1B , FIG. 1C and FIG. 1D The results of ligation fidelity analysis are shown in Figure 3. The results of ligation rate show that there is no significant difference in the ligation rate of the wild type enzyme for the correct and mismatched substrates, but the ligation rate of the two mutants for the mismatched substrates is significantly reduced. The results of ligation fidelity show that compared with the wild type enzyme, the K365A mutant increases the ligation fidelity to 1.7 times of the original, and the K367A mutant increases to 10.6 times of the original. These results prove that the two mutants have higher mismatch recognition ability and fidelity than the wild type enzyme.

[0024] The amino acid sequence of the wild type T4 DNA ligase (SEQ ID NO: 1) is as follows:

[0025] MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL.

[0026] The amino acid sequence of the T4 DNA ligase K365A mutant (SEQ ID NO: 2) is as follows:

[0027] MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYAFKEVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL.

[0028] The amino acid sequence of the T4 DNA ligase K367A mutant (SEQ ID NO: 3) is as follows:

[0029] MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFAEVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL.

[0030] The nucleotide sequence of the wild type of T4 DNA ligase (SEQ ID NO: 4) is as follows:

[0031]

[0032] The nucleotide sequence of the T4 DNA ligase K365A mutant (SEQ ID NO: 5) is as follows:

[0033]

[0034] The nucleotide sequence of the T4 DNA ligase K367A mutant (SEQ ID NO: 6) is as follows:

[0035]

Claims

1. Mutant of T4 DNA ligase with improved ligation fidelity, characterized in that, A T4 DNA ligase mutant obtained by mutating at least one of the lysine at position 365 or the lysine at position 367 of a wild-type T4 DNA ligase, wherein the sequence of the wild-type T4 DNA ligase is shown in SEQ ID NO:

1.

2. The mutant T4 DNA ligase with improved ligation fidelity according to claim 1, characterized in that, One or both of the lysine at position 365 or the lysine at position 367 is mutated to alanine or other amino acid.

3. The amino acid sequence of claim 1, wherein the mutant further comprises one or more substitutions and / or deletions and / or additions of amino acids, in addition to the amino acid mutation, and has substantially the same ligase activity and performance.

4. A gene encoding the T4 DNA ligase mutant of any one of claims 1-3.

5. An expression vector of the T4 DNA ligase mutant of any one of claims 1-3.

6. A host cell of the T4 DNA ligase mutant of any one of claims 1-3.

7. Use of the T4 DNA ligase mutant of any one of claims 1-3 in performing DNA-DNA, DNA-RNA, or RNA-RNA ligation.

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