PHI29 DNA polymerase mutant with increased thermal stability and its use

Description

FIELD

[0001] The present disclosure relates to a phi29 DNA polymerase mutant with improved thermal stability and application thereof.BACKGROUND

[0002] Phi29 DNA polymerase, belonging to the family B DNA polymerase, is a DNA polymerase derived from Bacillus subtilis phi29 phage. The crystal structure of phi29 DNA polymerase shows that phi29 DNA polymerase has two unique domains, i.e. TPR1 and TPR2 domains, in addition to conserved domains Palm, Thumb, Finger and Exo which are contained in common family B DNA polymerases, in which such a TPR2 domain takes part in forming a narrow channel surrounding the downstream DNA strand template, making the double-stranded DNA dissociated; meanwhile, the Palm, Thumb, TPR1 and TPR2 domains constitute a circular structure which tightly binds to the upstream double strands newly formed by template strand. Due to its structural characteristics, the phi29 DNA polymerase has a specific high processivity, strong strand displacement activity and 3'→5' exonuclease correction activity, thus commonly used in thermostatic amplification process, such as Rolling Circle Amplification (RCA) of micro amount of circular plasmids, Multiple Displacement Amplification (MDA) of genome and the like, and further applied to steps of library preparation through high-throughput sequencing, strand displacement amplification and the like.

[0003] Phi29 DNA polymerase is a mesophile enzyme, with a poor thermal stability, which can be inactivated by heating at 65°C for 10 minutes. In practice, the storage life of phi29 DNA polymerase product, effect of DNA amplification and sequencing and the like are often affected due to the poor thermal stability.

[0004] Regarding the improvement of thermal stability and optimization of amplification efficiency for phi29 DNA polymerase, the current research mainly focuses on aspects of 1) optimization of storage buffer or reaction buffer, such as adding some surfactants or compatible solutes, and 2) mutating the protein sequence of wild-type phi29 DNA polymerase or constructing a chimeric protein.

[0005] D1 (US2014322759) discloses a phi29 DNA polymerase characterized with SEO ID NO: 1 and the point mutation M97T, L123S, and / or E515A showing increased thermostability. D2 (US2011189659) discloses a phi29 DNA polymerase characterized with SEQ ID NO: 1 and the point mutation E515R / K showing increased thermostability. D3 (US2012034602) concerns the mutation E515P to increase thermostability of phi29 DNA polymerase. D4 (US2014094374) suggests the mutation L123K to increase thermostability of phi29 DNA polymerase.

[0006] Although improvement on thermal stability of phi29 DNA polymerase has been achieved to some extent for existing technology, the development on phi29 DNA polymerase with higher thermal stability is still needed. In one aspect, because mutation targeting thermal stability will affect function of other domains of polymerase thus further affecting downstream application, modification on phi29 DNA polymerase which is combined with different applications has practical significance. In another aspect, companies have a need to develop their own patented products to avoid the risk of patent infringement in view of commercial competition and restriction of patent rights.SUMMARY

[0007] The object of the present disclosure is to provide a phi29 DNA polymerase mutant with improved thermal stability and application thereof.

[0008] The present invention provides a protein, which is obtained by subjecting a phi29 DNA polymerase having SEQ ID NO: 1 to substitutions with three point mutations of point mutation A, point mutation B and point mutation C and keeping remaining amino acids unchanged, wherein the point mutation A is the mutation of amino acid residue Methionine (M) at position 97 of the phi29 DNA polymerase to Alanine (A) or Lysine (K); the point mutation B is the mutation of amino acid residue Leucine (L) at position 123 of the phi29 DNA polymerase to Lysine (K), Phenylalanine (F), Isoleucine (I) or Histidine (H); and the point mutation C is the mutation of amino acid residue Glutamic acid (E) at position 515 of the phi29 DNA polymerase to Proline (P), wherein the protein is a phi29 DNA polymerase mutant having increased thermal stability compared to the phi29 DNA polymerase having SEQ ID NO: 1.

[0009] Specifically, the protein in the present invention is any one selected from protein (1) to (6): (1) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Alanine (A), the mutation of amino acid residue Leucine (L) at position 123 to Histidine (H) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); (2) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Lysine (K), the mutation of amino acid residue Leucine (L) at position 123 to Isoleucine (I) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); (3) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Lysine (K), the mutation of amino acid residue Leucine (L) at position 123 to Phenylalanine (F) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); (4) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Lysine (K), the mutation of amino acid residue Leucine (L) at position 123 to Histidine (H) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); (5) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Lysine (K), the mutation of amino acid residue Leucine (L) at position 123 to Lysine (K) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); (6) a fusion protein obtained by ligating a tag at the N-terminus and / or the C-terminus of any protein of (1) to (5).

[0010] The present invention also provides a nucleic acid molecule encoding the protein as described in the above invention, an expression cassette containing the nucleic acid molecule, a recombinant vector containing the nucleic acid molecule, a recombinant bacterium containing the nucleic acid molecule, and a transgenic cell line containing the nucleic acid molecule.

[0011] The present invention further provides the protein as described in the above invention for use in any one of (a) to (g): (a) as a DNA polymerase; (b) catalyzing DNA replication and / or DNA amplification; (c) catalyzing rolling circle amplification and / or multiple-strand displacement amplification; (d) preparing a kit for catalyzing DNA replication and / or DNA amplification; (e) preparing a kit for catalyzing rolling circle amplification and / or multiple-strand displacement amplification; (f) DNA sequencing or RNA sequencing; and (g) preparing a kit for DNA sequencing or RNA sequencing.

[0012] The present invention further provides the nucleic acid molecule encoding the protein as described in the above invention, the expression cassette containing the nucleic acid molecule, the recombinant vector containing the nucleic acid molecule, the recombinant bacterium containing the nucleic acid molecule and the transgenic cell line containing the nucleic acid molecule, for use in any one of (h) to (k): (h) preparing a DNA polymerase; (i) preparing a kit for catalyzing DNA replication and / or DNA amplification; (j) preparing a kit for catalyzing rolling circle amplification and / or multiple-strand displacement amplification; and (k) preparing a kit for DNA sequencing or RNA sequencing. The present invention further provides a method of improving the stability of phi29 DNA polymerase, including subjecting a phi29 DNA polymerase having SEQ ID NO: 1 to substitutions with three point mutations of point mutation A, point mutation B and point mutation C and keeping remaining amino acids unchanged, wherein the point mutation A is the mutation of amino acid residue Methionine (M) at position 97 of the phi29 DNA polymerase to Alanine (A) or Lysine (K); the point mutation B is the mutation of amino acid residue Leucine (L) at position 123 of the phi29 DNA polymerase to Lysine (K), Phenylalanine (F), Isoleucine (I) or Histidine (H); and the point mutation C is the mutation of amino acid residue Glutamic acid (E) at position 515 of the phi29 DNA polymerase to Proline (P), wherein the protein is a phi29 DNA polymerase mutant having increased thermal stability compared to the phi29 DNA polymerase having SEQ ID NO: 1. DETAILED DESCRIPTION

[0013] The following examples are for better understanding of the present disclosure rather than limiting. Unless otherwise specified, the experimental methods in the following examples are conventional methods, and the test materials used in the following examples are purchased from conventional biochemical reagent companies. The quantitative experiments in the following examples are all set up in triplicate, with averaged results. Solvent in each solution or buffer solution in the following examples is water, unless otherwise specified.

[0014] pET28a (+) vector is from Novagen.

[0015] E. coli BL21 (DE3) is from TIANGEN, in a catalog number of CB105-02.

[0016] Storage buffer includes 10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5%(v / v) Tween ®< 20, 0.5% (v / v) NP-40 and 50% (v / v) Glycerol, with pH7.4 @ 25°C.

[0017] 141 RCA Primer in the examples is of a sequence: TCCTAAGACCGCTTGGCCTCCGACT (SEQ ID NO: 3).

[0018] 141Ad ssDNA in the examples is generated by BGI and is a circular single-strand library in a certain size range, without fixed sequences. Specifically, it is a random library consisting of four nucleotides (A / T / C / G), with a main band in a length of 200 to 300 bp.Example 1 Construction of recombinant bacterium and purification of protein1.1 Construction of recombinant vector

[0019] A wild-type recombinant vector (recombinant vector WT) was obtained by inserting the DNA molecule as shown in SEQ ID NO: 2 in the sequence listing between the NdeI and BamHI restriction sites of pET28a (+) vector. The DNA molecule as shown in SEQ ID NO: 2 in the sequence listing expresses the protein as shown in SEQ ID NO: 1 in the sequence listing, that is, the wild-type phi29 DNA polymerase represented by WT.

[0020] Different recombinant vectors were obtained by subjecting the recombinant vector WT as the original vector to point mutations in the presence of respective primer pairs in Table 1. Table 1Point mutationForward primerReverse primer97H97A97K123K123F123I123H 515P515G

[0021] The recombinant vector M97A differs with the recombinant vector WT only in that the nucleotides 289-291 of the DNA molecule shown in SEQ ID NO: 2 in the sequence listing are mutated from "ATG" to "GCG", with mutated DNA molecule encoding mutant M97A. The mutant M97A differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A).

[0022] The recombinant vector M97K differs with the recombinant vector WT only in that the nucleotides 289-291 of the DNA molecule shown in SEQ ID NO: 2 in the sequence listing are mutated from "ATG" to "AAA", with mutated DNA molecule encoding mutant M97K. The mutant M97K differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K).

[0023] The recombinant vector L123K differs with the recombinant vector WT only in that the nucleotides 367-369 of the DNA molecule shown in SEQ ID NO: 2 in the sequence listing are mutated from "CTG" to "AAA", with mutated DNA molecule encoding mutant L123K. The mutant L123K differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 123 is mutated from Leucine (L) to Lysine (K).

[0024] The recombinant vector E515G differs with the recombinant vector WT only in that the nucleotides 1543-1545 of the DNA molecule shown in SEQ ID NO: 2 in the sequence listing are mutated from "GAA" to "GGC", with mutated DNA molecule encoding mutant E515G. The mutant E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).

[0025] The recombinant vector M97A-L123I differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "GCG" and the nucleotides 367-369 are mutated from "CTG" to "ATT" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97A-L123I. The mutant M97A-L123I differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A) and the amino acid residue at position 123 is mutated from Leucine (L) to Isoleucine (I).

[0026] The recombinant vector M97A-L123H-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "GCG", the nucleotides 367-369 are mutated from "CTG" to "CAT" and the nucleotides 1543-1545 are mutated from "GAA" to "GGC" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97A-L123H-E515G. The mutant M97A-L123H-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).

[0027] The recombinant vector M97A-L123F-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "GCG", the nucleotides 367-369 are mutated from "CTG" to "TTT" and the nucleotides 1543-1545 are mutated from "GAA" to "GGC" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97A-L123F-E515G. The mutant M97A-L123F-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A), the amino acid residue at position 123 is mutated from Leucine (L) to Phenylalanine (F) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).

[0028] The recombinant vector M97A-L123H-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "GCG", the nucleotides 367-369 are mutated from "CTG" to "CAT" and the nucleotides 1543-1545 are mutated from "GAA" to "CCG" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97A-L123H-E515P. The mutant M97A-L123H-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).

[0029] The recombinant vector M97K-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "AAA" and the nucleotides 1543-1545 are mutated from "GAA" to "GGC" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-E515G. The mutant M97K-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).

[0030] The recombinant vector M97K-L123K-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "AAA", the nucleotides 367-369 are mutated from "CTG" to "AAA" and the nucleotides 1543-1545 are mutated from "GAA" to "CCG" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123K-E515P. The mutant M97K-L123K-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Lysine (K) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).

[0031] The recombinant vector M97K-L123F-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "AAA", the nucleotides 367-369 are mutated from "CTG" to "TTT" and the nucleotides 1543-1545 are mutated from "GAA" to "CCG" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123F-E5 15P. The mutant M97K-L123F-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Phenylalanine (F) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).

[0032] The recombinant vector M97K-L123I-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "AAA", the nucleotides 367-369 are mutated from "CTG" to "ATT" and the nucleotides 1543-1545 are mutated from "GAA" to "GGC" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123I-E515G. The mutant M97K-L123I-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Isoleucine (I) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).

[0033] The recombinant vector M97K-L123H-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "AAA", the nucleotides 367-369 are mutated from "CTG" to "CAT" and the nucleotides 1543-1545 are mutated from "GAA" to "GGC" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123H-E515G. The mutant M97K-L123H-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).

[0034] The recombinant vector M97K-L123I-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "AAA", the nucleotides 367-369 are mutated from "CTG" to "ATT" and the nucleotides 1543-1545 are mutated from "GAA" to "CCG" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123I-E515P. The mutant M97K-L123I-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Isoleucine (I) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).

[0035] The recombinant vector M97K-L123H-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "AAA", the nucleotides 367-369 are mutated from "CTG" to "CAT" and the nucleotides 1543-1545 are mutated from "GAA" to "CCG" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123H-E515P. The mutant M97K-L123H-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).

[0036] The recombinant vector M97H-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "CAT" and the nucleotides 1543-1545 are mutated from "GAA" to "GGC" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97H-E515G. The mutant M97H-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).

[0037] The recombinant vector M97H-L123I-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "CAT", the nucleotides 367-369 are mutated from "CTG" to "ATT" and the nucleotides 1543-1545 are mutated from "GAA" to "GGC" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97H-L123I-E515G. The mutant M97H-L123I-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Histidine (H), the amino acid residue at position 123 is mutated from Leucine (L) to Isoleucine (I) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).

[0038] The recombinant vector M97H-L123H-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from "ATG" to "CAT", the nucleotides 367-369 are mutated from "CTG" to "CAT" and the nucleotides 1543-1545 are mutated from "GAA" to "GGC" respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97H-L123H-E515G. The mutant M97H-L123H-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Histidine (H), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).1.2 Construction of recombinant bacterium

[0039] Different recombinant bacterium was obtained by introducing respective recombinant vector constructed in step 1.1 into E. coli BL21 (DE3).

[0040] Recombinant bacteria obtained were respectively named as recombinant bacterium WT, recombinant bacterium M97A, recombinant bacterium L123K, recombinant bacterium E515G, recombinant bacterium M97A-L123I, recombinant bacterium M97A-L123H-E515G, recombinant bacterium M97A-L123F-E515G, recombinant bacterium M97A-L123H-E515P, recombinant bacterium M97K-E515G, recombinant bacterium M97K-L123K-E515P, recombinant bacterium M97K-L123F-E515P, recombinant bacterium M97K-L123I-E515G, recombinant bacterium M97K-L123H-E515G, recombinant bacterium M97K-L123I-E515P, recombinant bacterium M97K-L123H-E515P, recombinant bacterium M97H-E515G, recombinant bacterium M97H-L123I-E515G and recombinant bacterium M97H-L123H-E515G, according to the principle corresponding to the name of recombinant vector.1.3 Induced expression of recombinant bacterium

[0041] The recombinant bacteria obtained in step 1.2 were respectively subjected to induction and purification, thus obtaining proteins fused to His 6 tag at N-terminus. Such proteins obtained were respectively named as a wild-type phi29 DNA polymerase with His 6 tag, a mutant M97A with His 6 tag, a mutant L123K with His 6 tag, a mutant E515G with His 6 tag, a mutant M97A-L123I with His 6 tag, a mutant M97A-L123H-E515G with His 6 tag, a mutant M97A-L123F-E515G with His 6 tag, a mutant M97A-L123H-E515P with His 6 tag, a mutant M97K-E515G with His 6 tag, a mutant M97K-L123K-E515P with His 6 tag, a mutant M97K-L123F-E515P with His 6 tag, a mutant M97K-L123I-E515G with His 6 tag, a mutant M97K-L123H-E515G with His 6 tag, a mutant M97K-L123I-E515P with His 6 tag, a mutant M97K-L123H-E515P with His 6 tag, a mutant M97H-E515G with His 6 tag, a mutant M97H-L123I-E515G with His 6 tag and a mutant M97H-L123H-E515G with His 6 tag, according to the principle corresponding to the name of recombinant bacterium.1.3.1 The induction process was conducted through the following specific steps:1.3.1.1 Activation of bacterium

[0042] The recombinant bacteria were inoculated into 3 ml liquid LB medium containing kanamycin, followed by culturing overnight.1.3.1.2 Transfer of bacterium solution

[0043] After the step 1.3.1.1, the obtained bacterium solution was transferred into 2 ml liquid LB medium containing kanamycin in volume of 1:100, followed by culturing under shaking at 37°C and 220 rpm to reach an OD 600nm value of 0.6, in which the OD 600nm value in a range of 0.4 to 0.8 is suitable in practice.1.3.1.3 Induction process

[0044] After the step 1.3.1.2, isopropyl-β-D-thiogalactoside (IPTG) was added to the system to be a final concentration of 0.5 mM, followed by culturing under shaking at 16°C and 220 rpm for 12 hours.1.3.1.4 Collection of bacterial cells

[0045] After the step 1.3.1.3, the system was centrifuged at 4°C and 8000 rpm for 5 minutes to collect bacterial cells.

[0046] 1.3.2 Bacterial cells were purified by using ÄKTAPure purification system from GE through the following specific steps.

[0047] 1.3.2.1 The bacterial cells obtained in step 1.3.1 were shakly mixed with the suspension buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM Imidazole, 5% Glycerol; pH 7.9 @ 25°C), ultrasonicated on ice, and centrifuged at 4°C and 12,000 rpm for 30 minutes, thus collecting the supernatant.

[0048] 1.3.2.2 The supernatant obtained in 1.3.2.1 was purified by using nickel column affinity chromatography (HisTrap FF 5ml prepacked column). Specifically, the supernatant was loaded after the column was balanced by 10 column volumes of Buffer A, after which the column was washed with 20 column volumes of Buffer A and eluted with 15 column volumes of eluent consisting of Buffer A and Buffer B, and the eluted solution with target protein was collected. During the elution, the volume fraction of Buffer B increased from 0% to 100% linearly, and the volume fraction of corresponding Buffer A decreased from 100% to 0% linearly.

[0049] Buffer A: 20 mM Tris-HCl, 500 mM NaCl, 20 mM Imidazole, 5% (v / v) Glycerol; pH 7.9@25°C.

[0050] Buffer B: 20 mM Tris-HCl, 500 mM NaCl, 500 mM Imidazole, 5% (v / v) Glycerol; pH 7.9@25°C.

[0051] 1.3.2.3 The eluted solution obtained in 1.3.2.2 was purified by using strong anion column chromatography (HiTrap Q HP 5ml prepacked column). Specifically, the eluted solution was loaded after the column was balanced by 10 column volumes of buffer mixture consisting of 59 volume % of Buffer A and 41 volume% of Buffer B. Collection of effluent was started after the protein peak occurred (that is, the UV detection value reached to be 20 mAu), and was stopped until the UV detection value dropped to 50 mAu again.

[0052] Buffer A: 20 mM Tris-HCl, 150 mM NaCl, 5% (v / v) Glycerol, pH 7.5@25°C.

[0053] Buffer B: 20 mM Tris-HCl, 1 M NaCl, 5% (v / v) Glycerol, pH 7.5@25°C.

[0054] 1.3.2.4 The effluent obtained in 1.3.2.3 was purified by using cation exchange chromatography (HiTrap SP HP prepacked column), thus obtaining a protein sample solution with a purity greater than 95%. Specifically, the effluent was loaded after the column was balanced by 10 column volumes of Buffer A, after which the column was washed with 15 column volumes of Buffer A and eluted with 10 column volumes of eluent consisting of Buffer A and Buffer B. During the elution, the volume fraction of Buffer B increased from 0% to 50% linearly, and the volume fraction of corresponding Buffer A decreased from 100% to 50% linearly. Collection of effluent containing target protein was started after the UV detection value reached to be 50 mAu and was stopped until the UV detection value dropped to 100 mAu again.

[0055] Buffer A: 20 mM Tris-HCl, 150 mM NaCl, 5% (v / v) Glycerol, pH 7.5@25°C.

[0056] Buffer B: 20 mM Tris-HCl, 1 M NaCl, 5% (v / v) Glycerol, pH 7.5@25°C.

[0057] 1.3.2.5 The target protein obtained in 1.3.2.4 was transferred to a dialysis bag, which was dialysed in the dialysis buffer overnight. The protein solution in the dialysis bag was collected and other components were added, thus obtaining a target potein solution containing 1 mg / ml of target potein. The other components in the target potein solution are 10 mM of Tris-HCl (pH7.4 @ 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5% (v / v) NP-40, 0.5% (v / v) Tween20 and 50% (v / v) Glycerol.

[0058] Dialysis buffer: 23.75 mM Tris-HCl (pH 7.4@25°C), 237.5 mM KCl, 2.375 mM DTT, 0.2375 mM EDTA and 5% (v / v) Glycerol.Example 2 Enzyme activity test of wild-type phi29 DNA polymerase and mutants

[0059] The taken target protein solution prepared in Example 1 (as an enzyme solution to be tested) was diluted to 5000 times by volume with the storage buffer, thoroughly mixted by a vortex shaker, and then stilled on ice for 5 minutes to obtain the solution to be tested.

[0060] 2.1 A pre-reaction system in a PCR tube after mixing was subjected to procedures in a PCR instrument: 95°C for 1 minute, 65°C for 1 minute and 40°C for 1 minute, with the hot lid set as a temperature of 102°C.

[0061] Pre-reaction system (80.8 µl): 50 mM Tris-HCl (pH 7.5), 4 mM DTT, 10 mM (NH 4 ) 2 SO 4 , 10 mM MgCl 2 , 50 nM dNTP Mixture, 2 pM 141 RCA Primer and 18 ng 141Ad ssDNA.

[0062] 2.2 After the step 2.1, the PCR tube was placed on ice when the temperature dropped to 4°C. For a test group, 1 µl of the solution to be tested was added; and for a negative control group, 1 µl of storage buffer was added. Both groups were mixed under shaking with a vortex shaker, centrifuged in a centrifuge for 5 seconds and then subjected to a procedure (i.e. heating at 30°C for 60 minutes) in the PCR instrument, with the hot lid set as a temperature of 65°C.

[0063] 2.3 After the step 2.2, 5 µl of 0.5M EDTA solution was added to terminate the reaction, and then mixed under shaking.

[0064] 2.4 The activity of the mixture obtained in 2.3 was assayed with Qubit ssDNA Assay Kit (Q10212, INVITROGEN) according to the instructions, and the concentration of DNA Nano ball (DNB) in the reaction product was detected by using Qubit fluorometer 3.0.Enzyme activity of enzyme solution to be tested = ΔDNB × 5000 ÷ 37.38

[0065] Note: ΔDNB is the difference of average concentration of reaction products in the reaction-terminated system between the test group and the negative control group, 5000 represents the dilution ratio and 37.38 represents the slope of function between enzyme activity and ΔDNB.

[0066] The results of enzyme activity of enzyme solution to be tested are shown in Table 2. Table 2PolymeraseAverage concentration of control groupAverage concentration of test groupΔDNB (ng / µl)Enzyme activity (U / µl)WT0.421.230.81108E515G0.400.760.3749.2L123K0.400.80.453.8M97A0.400.770.3850.5 Example 3 Thermal stability test of wild-type phi29 DNA polymerase and mutants

[0067] The taken target protein solution prepared in Example 1 was divided into two parts, which were respectively treated as follows.

[0068] First part: the target protein solution was placed in a metal bath preheated to 37°C for 10 minutes and centrifuged at 4°C and 13000 rpm for 1 minute to collect the supernatant. The supernatant obtained was diluted to 1000 times by volume with the storage buffer, thoroughly mixed by a vortex shaker, and then stilled on ice for 5 minutes to obtain the solution 1 to be tested.

[0069] Second part: the target protein solution was diluted to 5000 times by volume with the storage buffer, thoroughly mixed by a vortex shaker, and then stilled on ice for 5 minutes to obtain the solution 2 to be tested.

[0070] The solution 1 to be tested and the solution 2 to be tested were respectively detected according to steps 2.1 to 2.4 in Example 2. Enzyme activity without heat treatment U 1 = ΔDNB × 5000 ÷ 37.38 in which, ΔDNB is the difference of average concentration of reaction products in the reaction-terminated system between the test group (second part) and the negative control group; Enzyme activity with heat treatment U 2 = ΔDNB × 1000 ÷ 37.38 in which, ΔDNB is the difference of average concentration of reaction products in the reaction-terminated system between the test group (first part) and the negative control group; 5000 and 1000 represent the dilution ratio respectively, and 37.38 represents the slope of function between enzyme activity and ΔDNB; and Loss ratio of enzyme activity % = U 1 − U 2 ÷ U 1 × 100 % .

[0071] The results are shown in Table 3. Table 3Mutant NosDNB (ng / µl)ΔDNB (ng / µl)Enzyme activity (U / µl)Loss ratio of enzyme activity (%)NO.1WTwithout heat treatment1.230.81108.45100.0heat treatment0.410.000.00negative control0.42NO.2M97A-L123Iwithout heat treatment1.361.01134.4495.2heat treatment0.600.246.47negative control0.36NO.3M97A-L123H-E515Gwithout heat treatment0.740.3242.7859.2heat treatment1.070.6517.47negative control0.42NO.4M97A-L123F-ES15Gwithout heat treatment0.900.3951.6261.2heat treatment1.260.7520.03negative control0.51NO.5M97A-L123H-E515Pwithout heat treatment0.910.4053.5526.0heat treatment1.991.4839.61negative control0.51NO.6M97K-L123I-E515Pwithout heat treatment1.070.6384.2742.5heat treatment1.591.8148.48negative control0.44NO.7M97K-L123H-E515Pwithout heat treatment0.900.4560.8435.2heat treatment1.921.4739.43negative control0.44NO.8M97H-E515Gwithout heat treatment0.770.3547.2685.6heat treatment0.670.256.78negative control0.42NO.9M97H-L123I-E515Gwithout heat treatment0.870.4660.9186heat treatment0.740.328.53negative control0.42NO.10M97H-L123H-E515Gwithout heat treatment0.780.3749.0280.3heat treatment0.780.369.66negative control0.42NO.11M97Awithout heat treatment0.770.3850.5292.6heat treatment0.530.143.72negative control0.40NO.12L123Kwithout heat treatment0.800.4053.8596.8heat treatment0.460.061.70negative control0.40NO.13E515Gwithout heat treatment0.760.3749.2887.4heat treatment0.630.236.23negative control0.40NO.14M97Kwithout heat treatment1.481.07142.6397.5heat treatment0.540.133.55negative control0.41NO.15M97K-E515Gwithout heat treatment1.320.92123.4094.67heat treatment0.650.256.58negative control0.40NO.16M97K-L123K-E515Pwithout heat treatment0.850.4357.6058.77heat treatment1.310.8923.75negative control0.42NO.17M97K-L123F-E515Pwithout heat treatment0.860.4560.8053.03heat treatment1.481.0728.56negative control0.41NO.18MOY7K-L1231-E515Gwithout heat treatment1.380.95127.5486.28heat treatment1.080.6517.50negative control0.43NO.19M97K-L123H-E515Gwithout heat treatment1.451.05140.4185.80heat treatment1.150.7519.94negative control0.40 Example 4 Effect of wild-type phi29 DNA polymerase and mutants in DNA sequencing

[0072] Based on Example 3, several mutants with improved thermal stability were selected and detected for their effect on DNA sequencing through machine test on BGISEQ-500 sequencer according to the standard of BGISEQ-500 sequencer. All reagents used for the test are a complete set of PE50 V2.0 kit produced by BGI, E. coil Ad153 standard library produced by BGI and Qubit ssDNA Assay reagent produced by Invitrogen. The reagents used below are all included in the PE50 V2.0 kit, except for the library and Qubit ssDNA Assay reagent. The PE50 V2.0 reagent tank as described below only refers to the reagents used in the on-machine test.4.1 Preparation of DNBDNBs were prepared before on-machine test.

[0073] The DNBs were prepared through the specific steps as below.

[0074] 4.1.1 Each tube containing 20 µl DNB preparation buffer, 6 ng E. coil Ad153 standard library and molecular-grade water (for making up to a 40 µl system) after mixed via centrifugation was subjected to procedures in a PCR instrument: hot lid set as a temperature of 103°C; 95°C for 1 minute, 65°C for 1 minute and 40°C for 1 minute; holding at 4°C forever. After that, the tube was stilled on ice.

[0075] 4.1.2 After the step 4.1.1, 40 µl DNB polymerase mixture and 2.5 µl DNB polymerase II were added to each tube, mixed by a vortex shaker for 5 seconds, centrifuged briefly, and then placed in the PCR instrument at 30°C for 20 minutes, with the hot lid set as a temperature of 60°C.

[0076] 4.1.3 After the step 4.1.2, 20 µl DNB Stop Buffer was added to each tube, blew gently with a wide-mouth pipette and mixed for 20 times to terminate the reaction.

[0077] 4.1.4 After the step 4.1.3, the concentration of DNB generated was detected through the Qubit ssDNA Assay produced by Invitrogen according to the instruments, in which the concentration greater than 10 ng / µl is qualified.4.2 Loading of DNB

[0078] After preparation, the DNBs were loaded on a chip, that is, loading of DNB.

[0079] Loading of DNB was conducted according to the specific steps as below: A sample loading reagent plate V2.1 was taken to room temperature for melting, mixed under shaking, briefly centrifuged and placed on ice for use. DNB loading buffer II was taken, shaked for uniformity, briefly centrifuged and placed on ice for use. A chip and the sample loading reagent plate V2.1 were placed in the BGIDL-50. 35 µl of DNB loading buffer II was added to a PCR tube containing 100 µl DNB, gently mixed for 15 times with a wide-mouth pipette and arranged in a designated DNB area of the loading system. Loading process was initiated via the DNB loading program (Sample load 2.0), and the loaded chip was incubated at room temperature for 30 minutes and then stored at 2-8°C for use.4.3 On-machine test

[0080] The protein to be tested was subjected to on-machine sequencing on the BGI SEQ-500 sequencer by using a chip and a BGISEQ-500RS high-throughput sequencing reagent tank (PE50 V2.0). Before the on-machine sequencing, sequencing reagent tank II, dNTPs mixture (V3.0) and dNTPs mixture II (V2.0) were thawed and placed in a refrigerator or ice box at 4°C for use; and the DNA polymerase for sequencing was mixed under shaking and placed in an ice box for use. Specifically, a reagent for No. 5 well was formulated, that is, 1150 µl DNA polymerase mixture and 1150 µl dNTPs mixture (V3.0) were respectively transferred into the No. 5 well with a 1 ml pipette, and blew with the pipette for 10 to 15 times for uniformity; a reagent for No. 6 well was formulated, that is, 890 µl DNA polymerase mixture and 890 µl dNTPs mixture II (V2.0) were respectively transferred into the No. 6 well with a 1 ml pipette, and blew with the pipette for 10 to 15 times for uniformity; and a reagent for No. 14 well was formulated, that is, all reagent for the No. 14 well was taken with a 5 ml pipette, and 2.8 ml of the reagent for the No. 14 well and 400 µl phi29 polymerase mutant were mixed and transferred into this well. After that, those well-prepared reagent tanks were assembled. Finally, the on-machine sequencing was conducted, that is, initiating the sequencer, washing, placing the reagent tank in a designated position of the sequencer, pre-loading according to the operation sequence, assembling the chip prepared in step 4.2 after the pre-loading, filling in corresponding sequencing information, and starting the sequencing. After the completion of sequencing, the chip and reagent tank were removed, and the machine was washed.4.4 Data analysis

[0081] After the completion of sequencing, the analysis report was downloaded, and the performance of phi29 DNA polymerase mutants was evaluated according to previously specified criteria. For this, the wild-type phi29 DNA polymerase and its mutants were tested. Results in the example are shown in Table 4, in which the series number of mutants in Table 4 corresponds to that in Table 3. From the sequencing quality parameter AvgErrorRate%, mutants 97K-123I-515P and 97K-123H-515P are of the lowest value, followed by mutant 97A-123F-515G, and then mutants 97A-123H-515G and 97A-123H-515P, in contrast the wild-type phi29 DNA polymerase exhibits worst performance. In addition, the mutants in the example all have a value of parameter MappingRate% higher than that of wild-type phi29 DNA polymerase. Table 4Series number of mutantNO. 1NO. 3NO. 4NO. 5NO. 6NO. 7Sequencing quality parameterStandardrep.1rep.2rep.1rep.2rep.1rep.2rep.1rep.2rep.1rep.2rep.1rep.2ESR%≥808179.7384828483798188868888Q30%≥888688.4689898989888891919191Mapping Rate%≥9898.5398.869999999999999999100100AvgErrorRate%≤0.461.120.830.330.230.20.210.260.240.210.210.110.12ESR (effective spot rate) represents the ratio of total number of Reads to total number of DNBs on the chip; Q30 represents the ratio of bases with a quality value greater than 30 to the total base number; Mapping Rate represents the ratio of number of Reads mapped to the reference sequence to total Reads number; AvgError Rate represents average base error rate relative to the reference sequence. Industrial Application

[0082] The phi29 DNA polymerase in the prior art (i.e. wild-type phi29 DNA polymerase) has a poor thermal stability, thus resulting in a short shelf life of product and limited downstream application. The present disclosure has screened out several mutants with significantly improved thermal stability from large numbers of phi29 DNA polymerase mutants by using site-directed mutagenesis technology. On basis of the present disclosure, a mutant having a good effect can be further selected by subjecting the amino acids at the mutation sites of the present disclosure to a saturation mutation. Alternatively, on basis of the mutant of the present disclosure, similar effects can be achieved by mutating other amino acids except for the mutation sites included in the present disclosure. The mutant protein provided in the present disclosure has significantly improved thermal stability compared to the wild-type protein, which can greatly extend the shelf life of product and effectively improve the sequencing effect of the sequencing platform (such as, BGISEQ-500). Such mutant proteins can exist in the form of separately packaged DNA polymerase product or can be packaged in a DNA amplification kit or a DNA sequencing kit. The present disclosure also can be used in technical fields of food detection, virus detection, RNA detection, single cell sequencing and the like, as well as development of third or fourth generation sequencers.

Claims

1. A protein obtained by subjecting a phi29 DNA polymerase having SEQ ID NO: 1 to substitutions with three point mutations of point mutation A, point mutation B and point mutation C and keeping remaining amino acids unchanged, wherein the point mutation A is the mutation of amino acid residue Methionine (M) at position 97 of the phi29 DNA polymerase to Alanine (A) or Lysine (K); the point mutation B is the mutation of amino acid residue Leucine (L) at position 123 of the phi29 DNA polymerase to Lysine (K), Phenylalanine (F), Isoleucine (I) or Histidine (H); and the point mutation C is the mutation of amino acid residue Glutamic acid (E) at position 515 of the phi29 DNA polymerase to Proline (P), wherein the protein is a phi29 DNA polymerase mutant having increased thermal stability compared to the phi29 DNA polymerase having SEQ ID NO: 1.

2. The protein according to claim 1, wherein the protein is any one selected from (1) to (6), (1) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Alanine (A), the mutation of amino acid residue Leucine (L) at position 123 to Histidine (H) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); (2) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Lysine (K), the mutation of amino acid residue Leucine (L) at position 123 to Isoleucine (I) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); (3) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Lysine (K), the mutation of amino acid residue Leucine (L) at position 123 to Phenylalanine (F) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); (4) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Lysine (K), the mutation of amino acid residue Leucine (L) at position 123 to Histidine (H) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); (5) a protein obtained by subjecting the phi29 DNA polymerase to three point mutations and keeping remaining amino acids unchanged, wherein the three point mutations are the mutation of amino acid residue Methionine (M) at position 97 to Lysine (K), the mutation of amino acid residue Leucine (L) at position 123 to Lysine (K) and the mutation of amino acid residue Glutamic acid (E) at position 515 to Proline (P); and (6) a fusion protein obtained by ligating a tag at the N-terminus and / or the C-terminus of any protein of (1) to (5).

3. A nucleic acid molecule encoding the protein as defined in claim 1 or 2.

4. An expression cassette containing the nucleic acid molecule of claim 3.

5. A recombinant vector containing the nucleic acid molecule of claim 3.

6. A recombinant bacterium containing the nucleic acid molecule of claim 3.

7. A transgenic cell line containing the nucleic acid molecule of claim 3.

8. The protein according to claim 1 or 2 for use in any one of (a) to (g): (a) as a DNA polymerase; (b) catalyzing DNA replication and / or DNA amplification; (c) catalyzing rolling circle amplification and / or multiple-strand displacement amplification; (d) preparing a kit for catalyzing DNA replication and / or DNA amplification; (e) preparing a kit for catalyzing rolling circle amplification and / or multiple-strand displacement amplification; (f) DNA sequencing or RNA sequencing; and (g) preparing a kit for DNA sequencing or RNA sequencing.

9. The nucleic acid molecule encoding the protein as defined in claim 1 or 2, the expression cassette containing the nucleic acid molecule, the recombinant vector containing the nucleic acid molecule, the recombinant bacterium containing the nucleic acid molecule and the transgenic cell line containing the nucleic acid molecule, for use in any one of (h) to (k): (h) preparing a DNA polymerase; (i) preparing a kit for catalyzing DNA replication and / or DNA amplification; (j) preparing a kit for catalyzing rolling circle amplification and / or multiple-strand displacement amplification; and (k) preparing a kit for DNA sequencing or RNA sequencing.

10. A method of improving the stability of phi29 DNA polymerase, comprising subjecting a phi29 DNA polymerase having SEQ ID NO: 1 to substitutions with three point mutations of point mutation A, point mutation B and point mutation C and keeping remaining amino acids unchanged, wherein the point mutation A is the mutation of amino acid residue Methionine (M) at position 97 of the phi29 DNA polymerase to Alanine (A) or Lysine (K); the point mutation B is the mutation of amino acid residue Leucine (L) at position 123 of the phi29 DNA polymerase to Lysine (K), Phenylalanine (F), Isoleucine (I) or Histidine (H); and the point mutation C is the mutation of amino acid residue Glutamic acid (E) at position 515 of the phi29 DNA polymerase to Proline (P), wherein the protein is a phi29 DNA polymerase mutant having increased thermal stability compared to the phi29 DNA polymerase having SEQ ID NO: 1.