Stable and high-enzyme-activity phi29 dna polymerase, its coding gene and application

Abstract

Provided are a Phi29 DNA polymerase with stability and high enzyme activity, a coding gene thereof, and an application. The Phi29 DNA polymerase is obtained by substituting at least one of amino acid residues at positions 17, 96, 97, 99, 123, 140, 148, 158, 159, 171, 203, 204, 213, 217, 224, 250, 270, 309, 310, 320, 344, 345, 347, 369, 402, 416, 509, 515, and 524 of a DNA polymerase shown in SEQ ID NO: 2.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the stable and highly active Phi29 DNA polymerase, its encoding gene, and its applications. Background Technology

[0002] Phi29 DNA polymerase is a thermophilic DNA polymerase cloned from the Bacillus subtilis phage Phi29. It was expressed in E. coli using gene recombination technology and then purified and isolated after multiple purification processes. Due to its unique strand displacement activity, high fidelity, and continuous synthesis capability, Phi29 DNA polymerase is widely used in various isothermal amplification applications, such as RCA (rolling cycle amplification), MDA (multiple displacement amplification), and LAMP (loop-mediated isothermal amplification). In the DNB-SEQ sequencing platform, Phi29 DNA polymerase is mainly used for DNB making and two-strand amplification. However, both wild-type and commercially available Phi29 DNA polymerases exhibit poor stability, failing to meet kit requirements (e.g., wild-type Phi29 DNA polymerase has a stability of less than one year; its reaction rate is slow; and its sequencing quality is poor when used in DNB-SEQ). Therefore, improving the stability and / or enzyme activity of Phi29 DNA polymerase is of great significance. Summary of the Invention

[0003] The purpose of this invention is to provide a phi29 DNA polymerase with improved stability and / or enzyme activity (e.g., specific enzyme activity).

[0004] This invention first protects a protein, which may be C1 or C2):

[0005] C1) Replace at least one amino acid residue from positions 17, 96, 97, 99, 123, 140, 148, 158, 159, 171, 203, 204, 213, 217, 224, 250, 270, 309, 310, 320, 344, 345, 347, 369, 402, 416, 509, 515, and 524 in the amino acid sequence of the phi29 DNA polymerase to obtain a protein with DNA polymerase activity;

[0006] C2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in C1);

[0007] The amino acid sequence of the phi29 DNA polymerase is shown in SEQ ID NO: 2.

[0008] In the aforementioned protein, the T at position 17 can be replaced with P. The R at position 96 can be replaced with E, N, S, A, G, or K. The M at position 97 can be replaced with Y, S, R, Q, G, C, P, V, W, N, D, E, or T. The Q at position 99 can be replaced with V, E, or T. The L at position 123 can be replaced with Y, A, C, Q, M, N, or P. The T at position 140 can be replaced with K or H. The Y at position 148 can be replaced with P or E. The I at position 158 can be replaced with P. The T at position 159 can be replaced with A. The Q at position 171 can be replaced with E or K. The T at position 203 can be replaced with E. The T at position 204 can be replaced with K. The T at position 213 can be replaced with K. The G at position 217 can be replaced with K. The 224th bit Y can be replaced with E or K. The 250th bit V can be replaced with I. The 270th bit V can be replaced with R. The 309th bit F can be replaced with S. The 310th bit Y can be replaced with N or G. The 320th bit G can be replaced with H, E, D, or C. The 344th bit N can be replaced with R or K. The 345th bit V can be replaced with E. The 347th bit Y can be replaced with G. The 369th bit Y can be replaced with D or N. The 402nd bit K can be replaced with L. The 416th bit L can be replaced with A. The 509th bit V can be replaced with E. The 515th bit E can be replaced with W, T, S, R, N, I, F, H, Q, or T. The 524th bit I can be replaced with V.

[0009] The stability and / or specific enzyme activity of any of the above-mentioned proteins are higher than that of phi29 DNA polymerase.

[0010] The protein mentioned above can specifically be phi29 DNA polymerase T213K / L416A / V509E, phi29 DNA polymerase M97T / Y224K / E515S, or phi29 DNA polymerase R96S / L123P / Y224K / L416A / E515S.

[0011] The phi29 DNA polymerase T213K / L416A / V509E can be obtained by replacing T with K at position 213 from the N-terminus of SEQ ID NO: 2, L with A at position 416, and V with E at position 509.

[0012] The phi29 DNA polymerase M97T / Y224K / E515S can be obtained by replacing M at position 97 (starting from the N-terminus) of SEQ ID NO: 2 with T, Y at position 224 with K, and E at position 515 with S.

[0013] The phi29 DNA polymerase R96S / L123P / Y224K / L416A / E515S can be obtained by replacing R with S at position 96 from the N-terminus of SEQ ID NO: 2, L with P at position 123, Y with K at position 224, L with A at position 416, and E with S at position 515.

[0014] The protein mentioned above can specifically be any one of protein a1 to protein a70.

[0015] The protein a1 is obtained by replacing the T at the 17th position from the N end of SEQ ID NO: 2 with P.

[0016] The protein a2 is obtained by replacing M with Y at the 97th position from the N end of SEQ ID NO: 2.

[0017] The protein a3 is obtained by replacing the M at the 97th position from the N end of SEQ ID NO: 2 with S.

[0018] The protein a4 is obtained by replacing M with R at the 97th position from the N end of SEQ ID NO: 2.

[0019] The protein a5 is obtained by replacing M with Q at the 97th position from the N end of SEQ ID NO: 2.

[0020] The protein a6 is obtained by replacing M with G at the 97th position from the N end of SEQ ID NO: 2.

[0021] The protein a7 is obtained by replacing L with Y at position 123 from the N end of SEQ ID NO: 2.

[0022] The protein a8 is obtained by replacing the T at position 140 from the N end of SEQ ID NO: 2 with K.

[0023] The protein a9 is obtained by replacing the T at position 140 from the N end of SEQ ID NO: 2 with H.

[0024] The protein a10 is obtained by replacing the Y at position 148 from the N end of SEQ ID NO: 2 with P.

[0025] The protein a11 is obtained by replacing the Y at position 148 from the N end of SEQ ID NO: 2 with E.

[0026] The protein a12 is obtained by replacing the I at position 158 from the N end of SEQ ID NO: 2 with P.

[0027] The protein a13 is obtained by replacing the T at position 159 from the N end of SEQ ID NO: 2 with an A.

[0028] The protein a14 is obtained by replacing the T at position 203 from the N end of SEQ ID NO: 2 with E.

[0029] The protein a15 is obtained by replacing the Y at position 224 from the N end of SEQ ID NO: 2 with E.

[0030] The protein a16 is obtained by replacing the F at position 309 from the N end of SEQ ID NO: 2 with S.

[0031] The protein a17 is obtained by replacing the Y at the 310th position from the N end of SEQ ID NO: 2 with N.

[0032] The protein a18 is obtained by replacing the Y at position 310 from the N end of SEQ ID NO: 2 with G.

[0033] The protein a19 is obtained by replacing the G at position 320 from the N-terminus of SEQ ID NO: 2 with H.

[0034] The protein a20 is obtained by replacing the G at position 320 from the N-terminus of SEQ ID NO: 2 with E.

[0035] The protein a21 is obtained by replacing the G at position 320 from the N end of SEQ ID NO: 2 with a D.

[0036] The protein a22 is obtained by replacing the G at position 320 from the N end of SEQ ID NO: 2 with C.

[0037] The protein a23 is obtained by replacing the N at position 344 from the N end of SEQ ID NO: 2 with R.

[0038] The protein a24 is obtained by replacing the V at position 345 from the N end of SEQ ID NO: 2 with E.

[0039] The protein a25 is obtained by replacing the Y at position 347 from the N end of SEQ ID NO: 2 with G.

[0040] The protein a26 is obtained by replacing the V at position 509 from the N-terminus of SEQ ID NO: 2 with E.

[0041] The protein a27 is obtained by replacing the E at position 515 from the N-terminus of SEQ ID NO: 2 with W.

[0042] The protein a28 is obtained by replacing the E at position 515 from the N end of SEQ ID NO: 2 with a T.

[0043] The protein a29 is obtained by replacing the E at position 515 from the N end of SEQ ID NO: 2 with S.

[0044] The protein a30 is obtained by replacing the E at position 515 from the N end of SEQ ID NO: 2 with R.

[0045] The protein a31 is obtained by replacing the E at position 515 from the N end of SEQ ID NO: 2 with N.

[0046] The protein a32 is obtained by replacing the E at position 515 from the N end of SEQ ID NO: 2 with an I.

[0047] The protein a33 is obtained by replacing the E at position 515 from the N end of SEQ ID NO: 2 with F.

[0048] The protein a34 is obtained by replacing the R at the 96th position from the N end of SEQ ID NO: 2 with E.

[0049] The protein a35 is obtained by replacing the R at the 96th position from the N end of SEQ ID NO: 2 with N.

[0050] The protein a36 is obtained by replacing the R at the 96th position from the N end of SEQ ID NO: 2 with S.

[0051] The protein a37 is obtained by replacing the R at the 96th position from the N end of SEQ ID NO: 2 with A.

[0052] The protein a38 is obtained by replacing the R at the 96th position from the N end of SEQ ID NO: 2 with G.

[0053] The protein a39 is obtained by replacing the R at the 96th position from the N end of SEQ ID NO: 2 with K.

[0054] The protein a40 is obtained by replacing the M at the 97th position from the N end of SEQ ID NO: 2 with C.

[0055] The protein a41 is obtained by replacing M with P at the 97th position from the N end of SEQ ID NO: 2.

[0056] The protein a42 is obtained by replacing M with V at the 97th position from the N end of SEQ ID NO: 2.

[0057] The protein a43 is obtained by replacing M with W at the 97th position from the N end of SEQ ID NO: 2.

[0058] The protein a44 is obtained by replacing the M at the 97th position from the N end of SEQ ID NO: 2 with N.

[0059] The protein a45 is obtained by replacing the M at the 97th position from the N end of SEQ ID NO: 2 with D.

[0060] The protein a46 is obtained by replacing M with E at the 97th position from the N end of SEQ ID NO: 2.

[0061] The protein a47 is obtained by replacing Q with V at the 99th position from the N end of SEQ ID NO: 2.

[0062] The protein a48 is obtained by replacing the Q at the 99th position from the N end of SEQ ID NO: 2 with E.

[0063] The protein a49 is obtained by replacing the Q at the 99th position from the N end of SEQ ID NO: 2 with a T.

[0064] The protein a50 is obtained by replacing the L at position 123 from the N end of SEQ ID NO: 2 with A.

[0065] The protein a51 is obtained by replacing L with C at position 123 from the N end of SEQ ID NO: 2.

[0066] The protein a52 is obtained by replacing the L at position 123 from the N end of SEQ ID NO: 2 with Q.

[0067] The protein a53 is obtained by replacing L with M at position 123 from the N end of SEQ ID NO: 2.

[0068] The protein a54 is obtained by replacing the L at position 123 from the N end of SEQ ID NO: 2 with N.

[0069] The protein a55 is obtained by replacing the Q at position 171 from the N end of SEQ ID NO: 2 with E.

[0070] The protein a56 is obtained by replacing Q with K at position 171 from the N end of SEQ ID NO: 2.

[0071] The protein a57 is obtained by replacing the T at the 204th position from the N end of SEQ ID NO: 2 with K.

[0072] The protein a58 is obtained by replacing the T at position 213 from the N end of SEQ ID NO: 2 with K.

[0073] The protein a59 is obtained by replacing the G at position 217 from the N-terminus of SEQ ID NO: 2 with K.

[0074] The protein a60 is obtained by replacing the V at the 250th position from the N end of SEQ ID NO: 2 with I.

[0075] The protein a61 is obtained by replacing the V at position 270 from the N end of SEQ ID NO: 2 with R.

[0076] The protein a62 is obtained by replacing the N at position 344 from the N end of SEQ ID NO: 2 with K.

[0077] The protein a63 is obtained by replacing the Y at position 369 from the N end of SEQ ID NO: 2 with D.

[0078] The protein a64 is obtained by replacing the Y at position 369 from the N end of SEQ ID NO: 2 with N.

[0079] The protein a65 is obtained by replacing the K at position 402 from the N end of SEQ ID NO: 2 with L.

[0080] The protein a66 is obtained by replacing the L at the 416th position from the N end of SEQ ID NO: 2 with A.

[0081] The protein a67 is obtained by replacing the E at position 515 from the N-terminus of SEQ ID NO: 2 with H.

[0082] The protein a68 is obtained by replacing the E at position 515 from the N end of SEQ ID NO: 2 with Q.

[0083] The protein a69 is obtained by replacing the E at position 515 from the N end of SEQ ID NO: 2 with a T.

[0084] The protein a70 is obtained by replacing the I at position 524 from the N end of SEQ ID NO: 2 with V.

[0085] The protein mentioned above can specifically be any one of protein b1 to protein b70.

[0086] The protein b1 is obtained by replacing the T at the 37th position from the N end of SEQ ID NO:4 with P.

[0087] The protein b2 is obtained by replacing M with Y at position 117 from the N end of SEQ ID NO: 4.

[0088] The protein b3 is obtained by replacing the M at position 117 from the N end of SEQ ID NO: 4 with S.

[0089] The protein b4 is obtained by replacing M with R at position 117 from the N end of SEQ ID NO: 4.

[0090] The protein b5 is obtained by replacing the M at position 117 from the N end of SEQ ID NO: 4 with Q.

[0091] The protein b6 is obtained by replacing M with G at position 117 from the N end of SEQ ID NO: 4.

[0092] The protein b7 is obtained by replacing L with Y at position 143 from the N end of SEQ ID NO: 4.

[0093] The protein b8 is obtained by replacing the T at position 160 from the N end of SEQ ID NO:4 with K.

[0094] The protein b9 is obtained by replacing the T at position 160 from the N end of SEQ ID NO:4 with H.

[0095] The protein b10 is obtained by replacing the Y at position 168 from the N end of SEQ ID NO: 4 with P.

[0096] The protein b11 is obtained by replacing the Y at position 168 from the N end of SEQ ID NO:4 with E.

[0097] The protein b12 is obtained by replacing the I at position 178 from the N end of SEQ ID NO: 4 with P.

[0098] The protein b13 is obtained by replacing the T at position 179 from the N end of SEQ ID NO: 4 with an A.

[0099] The protein b14 is obtained by replacing the T at position 223 from the N end of SEQ ID NO: 4 with E.

[0100] The protein b15 is obtained by replacing the Y at position 244 from the N end of SEQ ID NO: 4 with E.

[0101] The protein b16 is obtained by replacing the F at position 329 from the N end of SEQ ID NO: 4 with S.

[0102] The protein b17 is obtained by replacing the Y at position 330 from the N end of SEQ ID NO:4 with N.

[0103] The protein b18 is obtained by replacing the Y at position 330 from the N end of SEQ ID NO:4 with G.

[0104] The protein b19 is obtained by replacing the G at position 340 from the N-terminus of SEQ ID NO:4 with H.

[0105] The protein b20 is obtained by replacing the G at position 340 from the N-terminus of SEQ ID NO:4 with E.

[0106] The protein b21 is obtained by replacing the G at position 340 from the N-terminus of SEQ ID NO:4 with a D.

[0107] The protein b22 is obtained by replacing the G at position 340 from the N-terminus of SEQ ID NO:4 with C.

[0108] The protein b23 is obtained by replacing the N at the 364th position from the N end of SEQ ID NO: 4 with R.

[0109] The protein b24 is obtained by replacing the V at position 365 from the N-terminus of SEQ ID NO: 4 with E.

[0110] The protein b25 is obtained by replacing the Y at position 367 from the N end of SEQ ID NO:4 with G.

[0111] The protein b26 is obtained by replacing the V at position 529 from the N-terminus of SEQ ID NO: 4 with E.

[0112] The protein b27 is obtained by replacing the E at position 535 from the N-terminus of SEQ ID NO: 4 with W.

[0113] The protein b28 is obtained by replacing the E at position 535 from the N end of SEQ ID NO: 4 with a T.

[0114] The protein b29 is obtained by replacing the E at position 535 from the N-terminus of SEQ ID NO: 4 with S.

[0115] The protein b30 is obtained by replacing the E at position 535 from the N-terminus of SEQ ID NO:4 with R.

[0116] The protein b31 is obtained by replacing the E at position 535 from the N end of SEQ ID NO: 4 with N.

[0117] The protein b32 is obtained by replacing the E at position 535 from the N-terminus of SEQ ID NO: 4 with an I.

[0118] The protein b33 is obtained by replacing the E at position 535 from the N-terminus of SEQ ID NO: 4 with F.

[0119] The protein b34 is obtained by replacing the R at position 116 from the N end of SEQ ID NO: 4 with E.

[0120] The protein b35 is obtained by replacing the R at position 116 from the N end of SEQ ID NO: 4 with N.

[0121] The protein b36 is obtained by replacing the R at position 116 from the N end of SEQ ID NO: 4 with S.

[0122] The protein b37 is obtained by replacing the R at position 116 from the N end of SEQ ID NO: 4 with an A.

[0123] The protein b38 is obtained by replacing the R at position 116 from the N end of SEQ ID NO:4 with G.

[0124] The protein b39 is obtained by replacing the R at position 116 from the N end of SEQ ID NO: 4 with K.

[0125] The protein b40 is obtained by replacing the M at position 117 from the N end of SEQ ID NO: 4 with C.

[0126] The protein b41 is obtained by replacing M with P at position 117 from the N end of SEQ ID NO: 4.

[0127] The protein b42 is obtained by replacing M with V at position 117 from the N end of SEQ ID NO: 4.

[0128] The protein b43 is obtained by replacing M with W at position 117 from the N end of SEQ ID NO: 4.

[0129] The protein b44 is obtained by replacing the M at position 117 from the N end of SEQ ID NO: 4 with N.

[0130] The protein b45 is obtained by replacing the M at position 117 from the N end of SEQ ID NO: 4 with a D.

[0131] The protein b46 is obtained by replacing M with E at position 117 from the N end of SEQ ID NO: 4.

[0132] The protein b47 is obtained by replacing Q with V at position 119 from the N end of SEQ ID NO: 4.

[0133] The protein b48 is obtained by replacing the Q at position 119 from the N-terminus of SEQ ID NO: 4 with an E.

[0134] The protein b49 is obtained by replacing the Q at position 119 from the N end of SEQ ID NO: 4 with a T.

[0135] The protein b50 is obtained by replacing the L at position 143 from the N end of SEQ ID NO: 4 with A.

[0136] The protein b51 is obtained by replacing the L at position 143 from the N end of SEQ ID NO: 4 with C.

[0137] The protein b52 is obtained by replacing the L at position 143 from the N end of SEQ ID NO: 4 with Q.

[0138] The protein b53 is obtained by replacing the L at position 143 from the N end of SEQ ID NO: 4 with M.

[0139] The protein b54 is obtained by replacing the L at position 143 from the N end of SEQ ID NO: 4 with N.

[0140] The protein b55 is obtained by replacing the Q at position 191 from the N-terminus of SEQ ID NO: 4 with an E.

[0141] The protein b56 is obtained by replacing Q with K at position 191 from the N end of SEQ ID NO: 4.

[0142] The protein b57 is obtained by replacing the T at position 224 from the N end of SEQ ID NO:4 with K.

[0143] The protein b58 is obtained by replacing the T at position 233 from the N end of SEQ ID NO:4 with K.

[0144] The protein b59 is obtained by replacing the G at position 237 from the N-terminus of SEQ ID NO:4 with K.

[0145] The protein b60 is obtained by replacing the V at position 270 from the N-terminus of SEQ ID NO: 4 with I.

[0146] The protein b61 is obtained by replacing the V at position 290 from the N end of SEQ ID NO: 4 with R.

[0147] The protein b62 is obtained by replacing the N at the 364th position from the N end of SEQ ID NO: 4 with K.

[0148] The protein b63 is obtained by replacing the Y at position 389 from the N end of SEQ ID NO: 4 with D.

[0149] The protein b64 is obtained by replacing the Y at position 389 from the N end of SEQ ID NO: 4 with N.

[0150] The protein b65 is obtained by replacing the K at position 422 from the N end of SEQ ID NO: 4 with L.

[0151] The protein b66 is obtained by replacing the L at the 436th position from the N end of SEQ ID NO: 4 with A.

[0152] The protein b67 is obtained by replacing the E at position 535 from the N-terminus of SEQ ID NO: 4 with H.

[0153] The protein b68 is obtained by replacing the E at position 535 from the N-terminus of SEQ ID NO: 4 with a Q.

[0154] The protein b69 is obtained by replacing the E at position 535 from the N end of SEQ ID NO: 4 with a T.

[0155] The protein b70 is obtained by replacing the I at position 544 from the N end of SEQ ID NO: 4 with V.

[0156] Nucleic acid molecules encoding any of the proteins described above are also within the scope of protection of this invention.

[0157] Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic cell lines containing the aforementioned nucleic acid molecules are also within the scope of protection of this invention.

[0158] The recombinant vector may be a recombinant plasmid obtained by inserting the nucleic acid molecule into an expression vector or a cloning vector. Specifically, the expression vector may be the vector pET28a(+).

[0159] The recombinant vector may specifically be one of the recombinant plasmids mentioned in the embodiments: pET28a-T17P, pET28a-M97Y, pET28a-M97S, pET28a-M97R, pET28a-M97Q, pET28a-M97G, pET28a-L123Y, pET28a-T140K, pET28a-T140H, pET28a-Y148P, pET28a-Y148E, pET28a-I158P, pET28a-T159A, and pET28a-T203E. Recombinant plasmids pET28a-Y224E, pET28a-F309S, pET28a-Y310N, pET28a-Y310G, pET28a-G320H, pET28a-G320E, pET28a-G320D, pET28a-G320C, pET28a-N344R, pET28a-V345E, pET28a-Y347G, pET28a-V509E, pET28a-E515W, pET28a-E515T, and pET28a-E 515S, recombinant plasmid pET28a-E515R, recombinant plasmid pET28a-E515N, recombinant plasmid pET28a-E515I, recombinant plasmid pET28a-E515F, recombinant plasmid pET28a-R96E, recombinant plasmid pET28a-R96N, recombinant plasmid pET28a-R96S, recombinant plasmid pET28a-R96A, recombinant plasmid pET28a-R96G, recombinant plasmid pET28a-R96K, recombinant plasmid pET28a-M97C, recombinant plasmid pET28a-M97P, recombinant plasmid pET28a-M97V, recombinant plasmid pET28a-M97W, recombinant plasmid pET28a-M97N. Recombinant plasmids pET28a-M97D, pET28a-M97E, pET28a-Q99V, pET28a-Q99E, pET28a-Q99T, pET28a-L123A, pET28a-L123C, pET28a-L123Q, pET28a-L123M, pET28a-L123N, pET28a-Q171E, pET28a-Q171K, pET28a-T204K, pET28a-T213K, and pET28a-G217K.Recombinant plasmids pET28a-V250I, pET28a-V270R, pET28a-N344K, pET28a-Y369D, pET28a-Y369N, pET28a-K402L, pET28a-L416A, pET28a-E515H, pET28a-E515Q, pET28a-E515T, or pET28a-I524V.

[0160] The recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant vector into the starting microorganism.

[0161] The originating microorganism may be Escherichia coli.

[0162] The specific Escherichia coli mentioned can be Escherichia coli BL21(DE3).

[0163] The use of any of the proteins or nucleic acid molecules described above in the preparation of DNA polymerase is also within the scope of protection of this invention.

[0164] In the above applications, the stability and / or specific activity of the DNA polymerase are higher than those of the phi29 DNA polymerase.

[0165] The application of any of the proteins or nucleic acid molecules described above in PCR amplification or sequencing is also within the scope of protection of this invention.

[0166] In the above applications, the PCR amplification can be two-stranded amplification, single-cell amplification, and / or plasmid amplification. The sequencing can be DNB SEQ sequencing.

[0167] The use of any of the proteins or nucleic acid molecules described above in the preparation of products for sequencing is also within the scope of protection of this invention.

[0168] In the above applications, the product may be a reagent kit.

[0169] Through extensive experimentation, the inventors of this invention performed site-directed mutagenesis on existing phi29 DNA polymerases and constructed combinatorial mutants using DNA shuffling and combinatorial mutant construction methods, resulting in 73 recombinant phi29 DNA polymerases with significantly improved stability and / or specific enzyme activity. These recombinant phi29 DNA polymerases not only exhibit improved thermostability but also enhanced polymerization activity and sustained synthesis capability. When the recombinant phi29 DNA polymerases prepared according to this invention are used for amplification or sequencing, DNA can be synthesized efficiently and continuously with high reaction efficiency. This invention has significant application value. Attached Figure Description

[0170] Figure 1 This is a schematic diagram of the structure of the carrier pET28a(+). Detailed Implementation

[0171] The following examples are provided to help to better understand the present invention, but are not intended to limit the invention.

[0172] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0173] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0174] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0175] The carrier pET28a(+) is a product of Novagen, and its structural diagram is shown below. Figure 1 .

[0176] Affinity A solution: an aqueous solution containing 20 mM Tris-HCl, 500 mM NaCl, 20 mM Imidazole and 62.5 g / L Glycerol, with a pH of 7.9.

[0177] The recombinant phi29 DNA polymerases in Examples 1, 2, and 3 are all single mutants of phi29 DNA polymerase, while the recombinant phi29 DNA polymerase in Example 4 is a combined mutant of phi29 DNA polymerase.

[0178] Example 1: Preparation of crude recombinant phi29 DNA polymerase

[0179] I. Construction of recombinant plasmid pET28a-WT

[0180] The small DNA fragment between the restriction endonuclease NdeI and BamHI recognition sequences of the vector pET28a(+) was replaced with the double-stranded DNA molecule shown in SEQ ID NO: 1, while all other sequences remained unchanged, to obtain the recombinant plasmid pET28a-WT.

[0181] The double-stranded DNA molecule shown in SEQ ID NO: 1 is the encoding gene of Phi29 DNA polymerase, and its encoded amino acid sequence is shown in SEQ ID NO: 2.

[0182] The recombinant plasmid pET28a-WT was sequenced. Sequencing results showed that in the recombinant plasmid pET28a-WT, the double-stranded DNA molecule shown in SEQ ID NO: 1 fused with the coding sequence of the His-tag tag (composed of 6 histidine residues) on the vector backbone, forming the fusion gene shown in SEQ ID NO: 3. This fusion gene expresses the recombinant Phi29 DNA polymerase shown in SEQ ID NO: 4 (named fusion protein 1), which possesses a His-tag tag.

[0183] II. Site-directed mutation of the gene encoding Phi29 DNA polymerase

[0184] 1. Prepare the site-directed mutagenesis PCR reaction system. The site-directed mutagenesis PCR reaction system is 25 μL, including 2.5 μL of 10×PfuReaction Buffer with Mg... 2+ 2 μL dNTP Mix (dATP, dTTP, dGTP and dCTP concentrations are all 2.5 mM), 25 ng recombinant plasmid pET28a-WT, 0.5 μL Pfu DNA Polymerase and mutation primers with added mutation sites.

[0185] Pfu DNA Polymerase is a product of Thermo Fisher Scientific, catalog number EP0501. 10×PfuReaction Buffer with Mg 2+ It is a component of Pfu DNA Polymerase.

[0186] The mutation primers with added mutation sites are shown in Table 1.

[0187] Table 1

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] 2. Take the site-directed mutagenesis PCR reaction system and perform PCR amplification to obtain the PCR amplification product.

[0194] The reaction program was as follows: 95℃ for 3 min; 95℃ for 30 s, 53℃ for 30 s, 68℃ for 8 min, 19 cycles; 68℃ for 10 min; stored at 4℃.

[0195] 3. Take the PCR amplification product, digest it with DpnI, transform it into Escherichia coli DH5α competent cells, then plate it on LB agar plates containing kanamycin, incubate it overnight at 37°C, pick single clones and extract plasmids.

[0196] 4. Sequencing the plasmids extracted in step 3. Based on the sequencing results, several recombinant plasmids with point mutations in the coding gene of Phi29 DNA polymerase were obtained, each encoding a different fusion protein (i.e., recombinant Phi29 DNA polymerase).

[0197] The fusion proteins encoded by some of the recombinant plasmids are shown in Table 2.

[0198] Table 2

[0199]

[0200]

[0201]

[0202] III. Preparation of crude recombinant phi29 DNA polymerase

[0203] The crude enzyme preparation method of recombinant phi29 DNA polymerase 1 is as follows:

[0204] 1. The recombinant plasmid pET28a-WT was transformed into Escherichia coli BL21(DE3) to obtain the recombinant bacteria, which was named BL21(DE3)-WT.

[0205] 2. Take a single BL21(DE3)-WT clone, inoculate it into 5 mL of LB liquid medium (containing 50 μg / mL kanamycin), and culture at 37℃ and 200 rpm for 12 h to obtain the culture solution.

[0206] 3. Take the cultured bacterial suspension and inoculate it into 1.5L LB liquid medium (containing 50μg / mL kanamycin) at a volume ratio of 1:100. Incubate at 37℃ with shaking at 200rpm until OD reaches the target value. 600nm The value was 0.6, then IPTG was added to make the concentration 0.5mM, and the mixture was cultured at 16℃ and 200rpm for 12h with shaking. After centrifugation at 4℃ and 8000rpm for 10min, the bacterial pellet was collected.

[0207] 4. After completing step 3, take the bacterial precipitate, add affinity solution A to resuspend it, incubate on ice for 30 min, and then sonicate it under ice-water bath conditions (using the Φ6 probe of Ningbo Xinzhi ultrasonic disruptor, ultrasonic power 40%, the cycle program is: disrupt for 2s, stop for 3s, for a total of 30 min), and then centrifuge at 4℃ and 15000rpm for 30 min, and collect the supernatant.

[0208] 5. After completing step 4, take the supernatant and perform rapid purification using affinity chromatography, followed by dialysis (the solute and concentration of the dialysis buffer are: 200mM KCl, 0.2mM EDTA, 5% Glyecrol and 20mM Tris-HCl; the solvent is water; the pH value is 7.5; the temperature is 25℃) to obtain the crude enzyme of recombinant phi29 DNA polymerase 1.

[0209] Following the steps described above, replace the recombinant plasmid pET28a-WT with the following recombinant plasmids respectively: pET28a-T17P, pET28a-M97Y, pET28a-M97S, pET28a-M97R, pET28a-M97Q, pET28a-M97G, pET28a-L123Y, pET28a-T140K, pET28a-T140H, pET28a-Y148P, pET28a-Y148E, pET28a-I158P, pET28a-T159A, and pET28a-T159A. T28a-T203E, recombinant plasmid pET28a-Y224E, recombinant plasmid pET28a-F309S, recombinant plasmid pET28a-Y310N, recombinant plasmid pET28a-Y310G, recombinant plasmid pET28a-G320H, recombinant plasmid pET28a-G320E, recombinant plasmid pET28a-G320D, recombinant plasmid pET28a-G320C, recombinant plasmid pET28a-N344R, recombinant plasmid pET28a-V345E, recombinant plasmid pET28a-Y347G, recombinant plasmid pET28a-V509E, recombinant plasmid pET28a-E515W, recombinant plasmid pET28a-E515T Recombinant plasmids pET28a-E515S, pET28a-E515R, pET28a-E515N, pET28a-E515I, pET28a-E515F, pET28a-R96E, pET28a-R96N, pET28a-R96S, pET28a-R96A, pET28a-R96G, pET28a-R96K, pET28a-M97C, pET28a-M97P, pET28a-M97V, and pET28a-M97W... Recombinant plasmids pET28a-M97N, pET28a-M97D, pET28a-M97E, pET28a-Q99V, pET28a-Q99E, pET28a-Q99T, pET28a-L123A, pET28a-L123C, pET28a-L123Q, pET28a-L123M, pET28a-L123N, pET28a-Q171E, pET28a-Q171K, pET28a-T204K, and pET28a-T213K.Recombinant plasmids pET28a-G217K, pET28a-V250I, pET28a-V270R, pET28a-N344K, pET28a-Y369D, pET28a-Y369N, pET28a-K402L, pET28a-L416A, pET28a-E515H, pET28a-E515Q, pET28a-E515T, and pET28a-I524V were used to sequentially obtain crude recombinant phi29 DNA polymerase 2 and crude recombinant phi29 DNA polymerase 71, while keeping other steps unchanged.

[0210] Stability detection of crude recombinant phi29 DNA polymerase prepared in Example 2 and Example 1.

[0211] The Tm values ​​of crude enzyme and dialysis buffer of the 71 recombinant phi29 DNA polymerases prepared in Example 1 were detected using a protein thermal shift assay kit (Life Technologies). Specifically, the program and reaction buffer were set up according to the protein thermal shift studies user guide; after the program was completed, the experimental results were input into the protein thermal shift software for analysis to obtain the Tm value of each sample.

[0212] Recombinant phi29 DNA polymerase 1 served as a positive control.

[0213] The dialysis buffer served as a negative control.

[0214] Each sample was repeated four times and the average value was taken. Some results are shown in Table 3. The results show that, compared with the crude enzyme of recombinant phi29 DNA polymerase 1, the Tm values ​​of crude enzymes of recombinant phi29 DNA polymerase 2 and recombinant phi29 DNA polymerase 34 were all increased to a certain extent, that is, the stability of crude enzymes of recombinant phi29 DNA polymerase 2 and recombinant phi29 DNA polymerase 34 was improved to a certain extent.

[0215] Table 3

[0216]

[0217]

[0218] Specific enzyme activity assay of crude enzyme in recombinant phi29 DNA polymerase prepared in Example 3 and Example 1.

[0219] The crude enzyme activity of the 71 recombinant phi29 DNA polymerases prepared in each Example 1 was determined:

[0220] 1. Take the crude enzyme of recombinant phi29 DNA polymerase and determine the protein concentration using the BCA kit; then dilute it with dialysis buffer to obtain a diluted solution of recombinant phi29 DNA polymerase with a concentration of 5 μg / mL.

[0221] The solutes and concentrations of the dialysis buffer were 20 mM Tris-HCl, 200 mM KCl, 2 mM DTT, 0.2 mM EDTA and 5% Glycerol; the solvent was water and the pH was 7.4.

[0222] 2. Prepare the reaction mixture. The reaction mixture is 80.8 μL and consists of DTT, (NH4)2SO4, MgCl2, dNTP mixture, RCA Primer (i.e., Ad153 make DNB primer; Invitrogen product, catalog number R082), 6 ng single-stranded circular DNA template 153Ad ssDNA, and pH 7.5, 50 mM Tris-HCl buffer. The concentrations of DTT, (NH4)2SO4, MgCl2, dNTP mixture, and RCA Primer in the reaction mixture are 4 mM, 10 mM, 10 mM, 50 nM, and 2 pM.

[0223] 3. Place the reaction mixture in a PCR instrument for primer-template hybridization. The program is as follows: 95℃ for 1 min, 65℃ for 1 min, 40℃ for 1 min, with the hot cap temperature set to 102℃. When the temperature reaches 4℃, remove the PCR tube and place it on ice. Add 1 μL of recombinant phi29 DNA polymerase dilution buffer, vortex to mix, briefly centrifuge for 5 seconds, and then place it in the PCR instrument for reaction. The reaction conditions are: 30℃ for 60 min, with the hot cap temperature set to 65℃. After the reaction is complete, add 5 μL of 0.5M EDTA solution to terminate the reaction, vortex to mix, and obtain the reaction product.

[0224] 4. Follow the instructions for the Qubit ssDNA assay kit and use a Qubit fluorometer 3.0 to detect the concentration of the reaction product obtained in step 3. Define 1U of enzyme activity as the amount of enzyme required to add 10 nmol dNTPs to DNB under the condition of reacting at 30℃ for 60 min. Further obtain the specific enzyme activity of the crude recombinant phi29 DNA polymerase.

[0225] Some results are shown in Table 4. The results indicate that, compared with the crude enzyme of recombinant phi29 DNA polymerase 1, the specific enzyme activities of the crude enzymes of recombinant phi29 DNA polymerase 3, recombinant phi29 DNA polymerase 4, recombinant phi29 DNA polymerase 5, and the crude enzyme-recombinant phi29 DNA polymerase 34 were all increased to some extent. That is, the DNA polymerase activities of the crude enzymes of recombinant phi29 DNA polymerase 3, recombinant phi29 DNA polymerase 4, recombinant phi29 DNA polymerase 5, and the crude enzyme-recombinant phi29 DNA polymerase 34 were all improved to some extent.

[0226] Table 4

[0227]

[0228]

[0229] Example 4: Obtaining, Detecting the Stability and Specific Enzyme Activity of Combinatorial Mutants of phi29 DNA Polymerase

[0230] I. Construction of recombinant phi29 DNA polymerase (i.e., a combinatorial mutant of phi29 DNA polymerase)

[0231] Based on the mutation sites provided in Example 1 and existing mutation sites disclosed in the literature, a combined mutant of phi29 DNA polymerase was constructed using the DNA shuffling method or the multi-site directed mutagenesis method.

[0232] The specific steps of the DNA shuffling method are as follows:

[0233] 1. Perform PCR amplification on the template to be shuffled (forward primer: 5'-CTGGTGCCGCGCGGCAGCCATATG-3', reverse primer: 5'-CTCGAATTCGGATCCTCACTTGA-3'), and then perform gel extraction and recovery.

[0234] 2. Perform the DNase I digestion reaction according to the steps in Table 5.

[0235] Table 5

[0236]

[0237] 3. After completing step 2, use M280 magnetic beads to recover the digested DNA fragments, wash twice with 75% (v / v) ethanol aqueous solution, and then dissolve with ddH2O.

[0238] 4. After completing step 3, perform shuffling recombination of the fragmented material using PCR.

[0239] The reaction system is shown in Table 6.

[0240] Table 6

[0241] reagents Volume (μL) <![CDATA[ddH2O]]> (21.5-DNA) DNA 0.25 / 0.5 / 1.0 10×pfu buffer 2.5 10mM dNTP 0.5 Pfu polymerase 0.5

[0242] The reaction program was as follows: 95℃ for 3 min; 95℃ for 30 s, 65℃ for 30 s, 72℃ for 1 min, 45 cycles; 72℃ for 7 min, and stored at 4℃.

[0243] 5. After completing step 4, use the recombined fragment as a template for secondary amplification and enrichment.

[0244] The reaction system is shown in Table 7.

[0245] Table 7

[0246] reagents Volume (μL) DNA 2.5 <![CDATA[ddH2O]]> 18.0 10×pfu buffer 2.5 10mM dNTP 0.5 10 μM Primer 1 (a mixture of the forward primers in Table 2) 0.5 10 μM Primer 2 (a mixture of reverse primers from Table 2) 0.5 2.5 U / μL Pfu polymerase 0.5

[0247] The reaction program was as follows: 95℃ for 3 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min 40 s, 60 cycles; 72℃ for 7 min.

[0248] Taking the construction of a mutant with 5 mutation sites as an example, the specific steps of the multi-site directed mutagenesis method are as follows:

[0249] 1. Primer design: The mutation site is designed in the middle of the primer, about 15 nt on each side. Each mutation site has a pair of reverse complementary primers.

[0250] 2. Prepare the reaction system. The reaction system is 25 μL, including 12.5 μL of 2×KAPA HiFi HS Ready Mix, 3.5 μL of 2 μM FW primer (5 strips in total, 0.7 μL each), 3.5 μL of 2 μM RE primer (5 strips in total, 0.7 μL each), 75 ng of template and water.

[0251] 3. Take the reaction system and perform PCR amplification.

[0252] The reaction program was as follows: 95℃ for 3 min; 98℃ for 20 s, 65℃ for 15 s, 72℃ for 7 min, 19 cycles; 72℃ for 10 min, and stored at 12℃.

[0253] 4. After completing step 3, add 1 μL of dpnI enzyme, digest at 37°C for 2 hours, then transform into E. coli DH5α competent cells, plate, and incubate overnight at 37°C. On the second day, pick a single colony, extract plasmids, and sequence them.

[0254] II. High-throughput screening of combinatorial mutants of phi29 DNA polymerase constructed in Example 1

[0255] The iCSR (isothermal compartmentalization self-replication) method was used for high-throughput screening of the combinatorial mutants of the phi29 DNA polymerase constructed in Example 1. Similar to CSR technology, the strand substitution function of phi29 DNA polymerase is used to replicate its own plasmid. The difference in DNA amplification amount among different mutants is used to characterize the strength of mutant activity. Through several rounds of screening, the high-activity mutants can be enriched. The specific steps are as follows:

[0256] 1. Primer design

[0257] The entire process requires three pairs of primers. Primer pair iCSR is used for amplification during the iCSR process (it needs to be thiolated at the 3' end to prevent digestion by intracellular exonucleases), and primer pair Insert and vector amplification primer pair are used for amplification of template and insert in the in-fusion reaction.

[0258] The iCSR primer pair consists of Primer 1: 5'-TTGAGGCCGTTGAGCACC-3' (3' end thiolated) and Primer 2: 5'-CCGGATATAGTTCCTCCTTTCAG-3' (3' end thiolated).

[0259] The primer pair Insert consists of Primer3: 5'-AATGTATAGCTGCGACTTTGAAACCA-3' and Primer4: 5'-TAGAGGCCCCAAGGGGTTAT-3'.

[0260] The vector amplification primer pair consists of Primer 5: 5'-ATAACCCCTTGGGGCCTCTA-3' and Primer 6: 5'-TGGTTTCAAAGTCGCAGCTATACAT-3'.

[0261] 2. Cell transformation and protein expression

[0262] The constructed mutant library was transformed into *E. coli* BL21 competent cells. Without plating, the cells, incubated at 37°C, were directly transferred to 2 mL of LB broth containing kanamycin and cultured overnight at 37°C. On the second day, the cells were again transferred to LB broth containing kanamycin at a 1:200 dilution and cultured at 37°C for 3 hours. Then, IPTG was added to a final concentration of 0.5 mM, and the cells were induced overnight at 16°C.

[0263] 3. Preparation of iCSR reaction system

[0264] 1) Prepare the reaction buffer. The reaction buffer is 2 mL, including 200 μL of 10×Phi29 reaction buffer, 40 μL of 500 μM Exo-resistant primer mix, 60 μL of 10 μM primer 1, 60 μL of 10 μM primer 2, 40 μL of 25 mM dNTP mix, and 1600 μL of NFH2O.

[0265] 2) Cell preparation

[0266] a. Mix 0.45 mL of 1×phi29 reaction buffer and 0.05 mL of 10 mg / mL lysozyme, and then preheat in a 30°C metal bath to obtain a cell lysis buffer.

[0267] b. OD value determination and final dilution volume calculation

[0268] To test the OD value of E. coli, the concentration was 8 × 10⁻⁶ when the OD value was 1. 8 Cells / mL estimation: Cell count = OD × 8 × 10 8 ×2=16×OD×10 8 Let the dilution volume be V mL.

[0269] Assuming the generated microdroplets have a diameter of approximately 21 μm, the volume of a single microdroplet is 5 pL. Assuming the flow rates of the bacterial channel and the buffer channel are the same, the volume of the bacterial channel is 2.5 pL.

[0270] λ = 16 × OD × 10 8 Cells / VmL × 2.5pL = 16 × 2.5 × OD × 10 8 ×10 -9 / V=4×OD / V

[0271] If λ = 0.2, then only 1% are double packages and 16% are single packages, so V = 20 × OD

[0272] c. Cell treatment

[0273] Collect induced *E. coli* cells, centrifuge at 12,000 rpm for 1 min, discard the supernatant, resuspend and wash twice with 1 mL of 1×Phi29 reaction buffer, then centrifuge at 12,000 rpm for 1 min, resuspend in 0.5 mL of cell lysis buffer, and incubate at 30°C and 300 rpm for 5 min. Centrifuge at 12,000 rpm for 1 min to recover the cells, then resuspend and dilute with V volumes of 1×Phi29 reaction buffer. Place on ice.

[0274] 4. Microdroplet preparation

[0275] The droplet diameter is controlled at around 20 μm. Note that the generated droplets need to be collected on ice, and about 500 μL of droplets should be collected.

[0276] 5. iCSR reaction

[0277] The droplets were divided into 30 μL portions per tube on ice and placed in PCR tubes. In this experiment, the gradient temperature settings of the PCR instrument were used to examine and screen mutants that could react at high temperatures. The experimental conditions were set to react at 37℃-55℃ for 2 h / 16 h, followed by heat inactivation of the phi29 DNA polymerase at 85℃ for 15 min.

[0278] 6. Delayed milk production

[0279] (1) Take the same number of phaseLock tubes and centrifuge at 16000g for 30s for pretreatment.

[0280] (2) Add an equal volume of PFO demulsifier to the PCR tube after the reaction is complete, mix thoroughly, transfer to a 1.5 mL EP tube, centrifuge at 14000 rpm for 10 min, then transfer all the liquid to a phaseLock tube, centrifuge at 16000 g for 5 min, take the upper layer liquid, and transfer it to a new PCR eight-tube.

[0281] 7. Enzyme digestion and qubit quantification

[0282] Take 9 μL of iCSR product into a new 8-tube PCR apparatus, add 0.5 μL of dpnI (to digest the template plasmid) and 0.5 μL of XbaI (to cut the amplified product into single copies), and digest at 37°C for 2 h. Then quantify using the qubit dsDNA HS assay kit (use 1 μL for quantification).

[0283] 8. Secondary amplification

[0284] Amplification was performed using the KAPA HiFi HotStart PCR Kit. Note that because no purification step was performed, Mg had already accumulated in the previous steps. 2+Therefore, the amount of reaction buffer needs to be adjusted, and note that readymix should not be used.

[0285] The reaction system consisted of 50 μL, including 8 μL of 5×HiFidelity buffer, 1.5 μL of 10 μM FW primer, 1.5 μL of 10 μM RE primer, 2 μL of template DNA (taken from a 3.7 tube), 1.5 μL of 10 mM dNTP mix, 34.5 μL of NFH2O, and 1 μL of HiFi Enzyme.

[0286] In addition, the amplification of the vector template can be performed normally using ReadyMix.

[0287] The reaction conditions were: 95℃ for 3 min, 98℃ for 20 s, 65℃ for 15 s, 72℃ for 2 min, 35 cycles; 72℃ for 10 min; and stored at 4℃.

[0288] 9. Glue recycling

[0289] Add 6× loading dye to the PCR product, then perform agarose gel electrophoresis. After gel excision, follow the procedures outlined in the gel extraction kit to recover the product. Quantify the recovered product.

[0290] 10. In-fusion reaction

[0291] According to the In-Fusion HD Cloning Kit instructions, the recommended dosage is 50-100 ng for insert lengths of 0.5-10 kb and 50-100 ng for vector lengths less than 10 kb. When there is only one insert, the recommended molar ratio of insert to vector is 2:1. Therefore, the reaction system of this invention is 10 μL, including 50 ng of purified PCR fragment, 78 ng of linearized vector, 2 μL of 5×In-fusion HD Enzyme mix, and NFH2O.

[0292] The reaction conditions were 50℃ for 15 min, followed by storage at 4℃.

[0293] 11. Transformation and Sequencing

[0294] The in-fusion product was directly transformed into KRX / BL21 competent cells (for higher transformation rates, DH5α cells can be transformed, plasmids extracted, and then transformed into BL21 cells), and then directly into LB broth containing kanamycin. On the second day, the bacterial culture was collected for induction expression and the next round of screening. After the second round of screening was completed, the cells were plated for sequencing.

[0295] After the above steps, four combined mutants of phi29 DNA polymerase with good activity were obtained, namely phi29 DNA polymerase T213K / L416A / V509E, phi29 DNA polymerase M97T / Y224K / E515S, phi29 DNA polymerase L123Q / T159A / Y347G and phi29 DNA polymerase R96S / L123P / Y224K / L416A / E515S.

[0296] The only difference between the phi29 DNA polymerase T213K / L416A / V509E and the Phi29 DNA polymerase shown in SEQ ID NO: 2 is that the latter has T replaced by K at position 213, L replaced by A at position 416, and V replaced by E at position 509.

[0297] The only difference between the phi29 DNA polymerase M97T / Y224K / E515S and the Phi29 DNA polymerase shown in SEQ ID NO: 2 is that the latter replaces M at position 97 with T, Y at position 224 with K, and E at position 515 with S.

[0298] The only difference between the phi29 DNA polymerase L123Q / T159A / Y347G and the Phi29 DNA polymerase shown in SEQ ID NO: 2 is that the latter replaces L at position 123 with Q, T at position 159 with A, and Y at position 347 with G.

[0299] The only difference between the phi29 DNA polymerase R96S / L123P / Y224K / L416A / E515S and the Phi29 DNA polymerase shown in SEQ ID NO: 2 is that the latter replaces R at position 96 with S, L at position 123 with P, Y at position 224 with K, L at position 416 with A, and E at position 515 with S.

[0300] III. Obtaining Combinatorial Mutants of phi29 DNA Polymerase

[0301] Prepare the Phi29 DNA polymerase, phi29 DNA polymerase T213K / L416A / V509E, phi29 DNA polymerase M97T / Y224K / E515S, phi29 DNA polymerase L123Q / T159A / Y347G and phi29 DNA polymerase R96S / L123P / Y224K / L416A / E515S shown in SEQ ID NO: 2.

[0302] IV. Stability testing of phi29 DNA polymerase combination mutants

[0303] The Tm values ​​of phi29 DNA polymerases T213K / L416A / V509E, M97T / Y224K / E515S, L123Q / T159A / Y347G, and R96S / L123P / Y224K / L416A / E515S were detected according to the method in Example 2.

[0304] The test results are shown in Table 8. The results indicate that, compared with the Phi29 DNA polymerase shown in SEQ ID NO: 2, the Tm values ​​of the combined mutants of the three phi29 DNA polymerases were all significantly increased, meaning that the stability of the combined mutants of the three phi29 DNA polymerases was significantly improved.

[0305] Table 8

[0306] Tm (°C) The Phi29 DNA polymerase shown in SEQ ID NO: 2 48.5 phi29 DNA polymerase T213K / L416A / V509E 49.4 phi29 DNA polymerase M97T / Y224K / E515S 50.8 phi29 DNA polymerase L123Q / T159A / Y347G 46.1 phi29 DNA polymerase R96S / L123P / Y224K / L416A / E515S 51.6

[0307] V. Specific enzyme activity assay of phi29 DNA polymerase combinatorial mutants

[0308] Following the method in Example 3, the specific enzyme activities of phi29 DNA polymerases T213K / L416A / V509E, M97T / Y224K / E515S, L123Q / T159A / Y347G, and R96S / L123P / Y224K / L416A / E515S were detected.

[0309] The test results are shown in Table 9. The results indicate that, compared with the Phi29 DNA polymerase shown in SEQ ID NO: 2, the specific enzyme activity of the combined mutant of the two phi29 DNA polymerases was significantly increased, meaning that the DNA polymerase activity of the combined mutant of the two phi29 DNA polymerases was significantly improved.

[0310] Table 9

[0311] Enzyme activity (U / μg) The Phi29 DNA polymerase shown in SEQ ID NO: 2 43 phi29 DNA polymerase T213K / L416A / V509E 52 phi29 DNA polymerase M97T / Y224K / E515S 55 phi29 DNA polymerase L123Q / T159A / Y347G 9 phi29 DNA polymerase R96S / L123P / Y224K / L416A / E515S 28

[0312] Therefore, it can be seen that phi29 DNA polymerases T213K / L416A / V509E and phi29 DNA polymerases M97T / Y224K / E515S have good stability and high specific activity, and are the most effective; phi29 DNA polymerases R96S / L123P / Y224K / L416A / E515S have good stability but slightly lower specific activity; phi29 DNA polymerases L123Q / T159A / Y347G have both poor stability and low specific activity.

[0313] Industrial applications

[0314] Compared with existing Phi29 DNA polymerases, this invention prepares 73 recombinant Phi29 DNA polymerases with significantly improved stability and / or specific enzyme activity. These recombinant Phi29 DNA polymerases not only exhibit improved thermostability but also enhanced polymerization activity and sustained synthesis capability. When amplification or sequencing is performed using the recombinant Phi29 DNA polymerases prepared according to this invention, DNA can be synthesized efficiently and continuously, resulting in high reaction efficiency. This invention has significant application value. SEQUENCE LISTING <110> Shenzhen BGI Life Science Research Institute <120> The stable and highly active Phi29 DNA polymerase, its encoding gene, and its applications <130> FSIOC1224718PCN <140> CN202080101598.7 <141> 2020-10-10 <150> CN202010523223.1 <151> 2020-06-10 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1728 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(1728) <223> CDS <400> 1 atgaagcata tgccgcgcaa aatgtatagc tgcgactttg aaaccaccac caaagtggaa gattgccgcg tttggggcgta tggctatatg aacatcgaag accacagcga atacaaaatt ggcaacagcc tggatgaatt tatggcgtgg gtgctgaaag ttcaggcgga tctgtatttt cacaacctga aatttgacgg cgcgttcatt attaactggc tggaacgcaa cggctttaaa 300. tggagcgcgg atggcttacc gaacacctat aacaccatta ttagccgcat gggccagtgg 360. the father of the father of the father of the father of the father of the father of the father of the father of the father of the father of the father of the father of the father gatagcctga agaaactgcc gtttccggtg aaaaaaatcg cgaaggactt taaactgacc gtgctgaaag gcgatattga ctaccataaa gaacgcccgg tgggctaa aattaccccg gaggaatatg cgtacatcaa gaacgacatc cagattattg cggaagcgct gctgattcag tttaaacagg gcctggatcg tatgaccgcg ggtagcgata gcctgaaagg ctttaaggac attack ccaagaagtt caagaagtg tttccgaccc tgagcctggg cctggataaa gaagtgcgct atgcgtatcg cggtggcttt acctggctga acgatcgctt taaggaaaag 720 gaaattggcg aaggcatggt gtttgatgtg aacagcctgt atccggcgca gatgtatagc 780 cgcctgctgc cgtatggtga accgattgtg tttgaaggca agtatgtgtg ggatgaagat 840 tatccgctgc acattcagca tattcgctgc gaattcgaac tgaaggaagg ctatattccg 900 accattcaga ttaaacgcag ccgcttttat aaaggcaacg agtacctgaa aagcagcggc 960 ggcgaaattg cggatctgtg gctgagcaac gtggatctgg aactgatgaa agaacactac 1020 gatctgtaca acgtggaata tatcagcggc ctgaaattta aagcgaccac cggcctgttt 1080 aagacttta tcgacaagtg gacctacatt aaaaccacca gcgaaggcgc gattaaacag 1140 ctggcgaaac tgatgctgaa cagcctgtat ggcaaatttg cgagcaaccc ggatgttacc 1200 ggcaaagtgc cgtatctgaa agaaaacggc gcgctgggct ttcgtttagg cgaagaggaa 1260 accaaagatc cggtgtatac cccgatgggc gtgtttatta ccgcgtgggc gcgctatacc 1320 accattaccg cggcgcaggc gtgttatgat cgcattatct attgcgatac cgatagcatt 1380 catctgaccg gcaccgaaat tccggatgtg atcaaagata ttgtggaccc gaaaaaactg 1440 ggctattggg cgcatgaaag cacctttaaa cgcgcgaaat atctgcgcca gaaaacctat 1500 atccaggaca tctacatgaa agaggtggat ggcaaactgg ttgaaggcag cccggatgat 1560 tataccgata ttaagttcag cgtgaaatgc gcgggcatga ccgataaaat taagaaggaa 1620 gtgaccttcg agaactttaa agtgggcttt agccgcaaaa tgaaaccgaa accggttcag 1680 gtgcctggcg gtgttgttct ggtggatgat accttcacca tcaagtga 1728 <210> 2 <211> 575 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 2 Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr 1 5 10 15 Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25 30 Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met 35 40 45 Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asn Leu Lys 50 55 60 Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys 65 70 75 80 Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95 Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110 Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125 Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140 Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro 145 150 155 160 Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170 175 Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser 180 185 190 Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205 Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220 Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys 225 230 235 240 Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255 Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270 Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285 Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295 300 Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly 305 310 315 320 Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335 Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350 Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365 Tyr Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380 Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr 385 390 395 400 Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410 415 Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe 420 425 430 Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445 Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460 Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu 465 470 475 480 Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495 Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510 Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515,520,525 Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Glu Val Thr Phe Glu 530 535 540 Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln 545 550 555 560 Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 565,570,575 <210> 3 <211> 1788 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 3 atgggcagca gccatcatca tcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgaagcata tgccgcgcaa atgtatagc tgcgactttg aaaccaccac caagtggaa 120 gattgccgcg tttggcgta tggctatatg aacatcgaag accacagcga atacaaaatt 180 ggcacagcc tggatgaatt tatggcgtgg gtgctgaaag ttcaggcgga tctgtatttt 240 cacaacctga aatttgacgg cgcgttcatt atttactggc tggaacgca cggctttaaa 300 tggagcgcgg atggcttacc gaacacctat aacaccatta ttagccgcat gggccagtgg 360 tatatgattg atatctgcct gggcttaaa gggcaacgca agatcatac cgtgatctat 420 gatagcctga agaactgcc gttccggtg aaaaaaatcg cgaaggactt taaactgacc 480 gtgctgaaag gcgatattga ctaccataaa gaacgcccgg tgggctataa aattaccccg 540 gaggaatg cgtacatca gaacgacatc cagattattg cggaagcgct gctgattcag 600 tttaaacagg gcctggatcg tatgaccgg ggtagcgata gcctgaagg ctttaaggac 660 attatcacca ccaagagtt cagaaagtg tttccgaccc tgagcctggg cctggataaa 720 gaagtgcgct atgcgtacg cggtggcttt acctggctga acgatcgctt taggaaaag 780 gaaattggcg aaggcatggt gttgatgtg aacagcctgt atccggcgca gatgtatagc 840 cgcctgctgc cgtatggtga accgattgtg tttgaggca agtatgtgtg ggatgaagat 900 tatccgctgc acatcagca tattcgctgc gattcgaac tgaaggagg ctatattccg 960 accattcaga ttaaacgcag ccgcttttat aaggcaacg agtacctgaa aagcagcggc 1020 ggcgaaattg cggatctgtg gctgagcaac gtggatctgg aactgatgaa agaacactac 1080 gatctgtaca acgtggaata tatcagcggc ctgaaattta aagcgaccac cggcctgttt 1140 aaggacttta tcgacaagtg gacctacatt aaaaccacca gcgaaggcgc gattaaacag 1200 ctggcgaaac tgatgctgaa cagcctgtat ggcaaatttg cgagcaaccc ggatgttacc 1260 ggcaaagtgc cgtatctgaa agaaaacggc gcgctgggct ttcgtttagg cgaagaggaa 1320 accaaagatc cggtgtatac cccgatgggc gtgtttatta ccgcgtgggc gcgctatacc 1380 accattaccg cggcgcaggc gtgttatgat cgcattatct attgcgatac cgatagcatt 1440 catctgaccg gcaccgaaat tccggatgtg atcaaagata ttgtggaccc gaaaaaactg 1500 ggctattggg cgcatgaaag cacctttaaa cgcgcgaaat atctgcgcca gaaaacctat 1560 atccaggaca tctacatgaa agaggtggat ggcaaactgg ttgaaggcag cccggatgat 1620 tataccgata ttaagttcag cgtgaaatgc gcgggcatga ccgataaaat taagaaggaa 1680 gtgaccttcg agaactttaa agtgggcttt agccgcaaaa tgaaaccgaa accggttcag 1740 gtgcctggcg gtgttgttct ggtggatgat accttcacca tcaagtga 1788 <210> 4 <211> 595 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 4 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp 20 25 30 Phe Glu Thr Thr Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly 35 40 45 Tyr Met Asn Ile Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu 50 55 60 Asp Glu Phe Met Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe 65 70 75 80 His Asn Leu Lys Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg 85 90 95 Asn Gly Phe Lys Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr 100 105 110 Ile Ile Ser Arg Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly 115 120 125 Tyr Lys Gly Lys Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys 130 135 140 Lys Leu Pro Phe Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr 145 150 155 160 Val Leu Lys Gly Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr 165 170 175 Lys Ile Thr Pro Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile 180 185 190 Ile Ala Glu Ala Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met 195 200 205 Thr Ala Gly Ser Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr 210 215 220 Lys Lys Phe Lys Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys 225 230 235 240 Glu Val Arg Tyr Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg 245 250 255 Phe Lys Glu Lys Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser 260 265 270 Leu Tyr Pro Ala Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro 275 280 285 Ile Val Phe Glu Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His 290 295 300 Ile Gln His Ile Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro 305 310 315 320 Thr Ile Gln Ile Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu 325 330 335 Lys Ser Ser Gly Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp 340 345 350 Leu Glu Leu Met Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile 355 360 365 Ser Gly Leu Lys Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile 370 375 380 Asp Lys Trp Thr Tyr Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln 385 390 395 400 Leu Ala Lys Leu Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn 405 410 415 Pro Asp Val Thr Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu 420 425 430 Gly Phe Arg Leu Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro 435 440 445 Met Gly Val Phe Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala 450 455 460 Ala Gln Ala Cys Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile 465 470 475 480 His Leu Thr Gly Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp 485 490 495 Pro Lys Lys Leu Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala 500 505 510 Lys Tyr Leu Arg Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu 515 520 525 Val Asp Gly Lys Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile 530 535 540 Lys Phe Ser Val Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu 545 550 555 560 Val Thr Phe Glu Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro 565 570 575 Lys Pro Val Gln Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe 580 585 590 Thr Ile Lys 595

Claims

1. A protein, C1 or C2), characterized by: The protein described in C1 is: phi29 DNA polymerase M97Y is the protein obtained by replacing M with Y at the 97th position from the N end of SEQ ID NO:

2. phi29 DNA polymerase M97C is a protein obtained by replacing M with C at the 97th position from the N end of SEQ ID NO:

2. phi29 DNA polymerase M97P is the protein obtained by replacing M with P at the 97th position from the N end of SEQ ID NO:

2. phi29 DNA polymerase M97V is the protein obtained by replacing M with V at the 97th position from the N end of SEQ ID NO:

2. phi29 DNA polymerase M97W is the protein obtained by replacing M with W at the 97th position from the N end of SEQ ID NO:

2. phi29 DNA polymerase M97N is the protein obtained by replacing the M at the 97th position from the N end of SEQ ID NO: 2 with N; phi29 DNA polymerase Y224E is a protein obtained by replacing the Y at position 224 from the N-terminus of SEQ ID NO: 2 with E. phi29 DNA polymerase L123C is a protein obtained by replacing L with C at position 123 from the N end of SEQ ID NO:

2. phi29 DNA polymerase L123N, which is the protein obtained by replacing the L at position 123 from the N-terminus of SEQ ID NO: 2 with N; or The phi29 DNA polymerase M97T / Y224K / E515S is a protein obtained by replacing M with T at position 97 from the N end of SEQ ID NO: 2, replacing Y with K at position 224, and replacing E with S at position 515. C2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in C1); The amino acid sequence of the phi29 DNA polymerase is shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the protein as described in claim 1.

3. An expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule as described in claim 2.

4. The recombinant vector as described in claim 3, characterized in that: The recombinant vector is a recombinant plasmid obtained by inserting the nucleic acid molecule into an expression vector or cloning vector.

5. The recombinant microorganism as described in claim 3, characterized in that: The recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant vector of claim 3 into the starting microorganism.

6. A transgenic cell line containing the nucleic acid molecule as described in claim 2.

7. The use of the protein as described in claim 1 or the nucleic acid molecule as described in claim 2 in PCR amplification or sequencing.

8. The application as described in claim 7, characterized in that: The PCR amplification includes two-stranded amplification, single-cell amplification, and / or plasmid amplification. The sequencing was DNB SEQ sequencing.

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