CN112079903B - Mutant of mismatching binding protein and coding gene thereof - Google Patents

Abstract

The invention provides a mismatch binding protein eMuts, the amino acid sequence of which comprises the amino acid sequence shown in SEQ ID NO. 52 and has at least one mutation selected from the group consisting of: 1) Serine at positions 120 and 151 were mutated to cysteine; 2) Leucine at position 157 and glycine at position 233 were mutated to cysteine; 3) Glutamic acid at position 451 and valine at position 465 are mutated to cysteine; 4) The 609 th methionine and the 723 th threonine are mutated into cysteine; or 5) serine at position 120, leucine at position 157, glutamic acid at position 451, and methionine at position 609 are all mutated to cysteine. The mismatch binding protein provided by the invention has higher thermal stability, and can stably and specifically identify and bind to DNA containing an error sequence, thereby improving the accuracy of the DNA. The invention also provides host cells comprising a gene encoding the mismatch binding protein and methods of producing the mismatch binding protein using the same.

Description

Mutant of mismatching binding protein and coding gene thereof

Technical Field

The invention relates to the field of biotechnology. In particular, the invention relates to mutants of mismatch binding protein (eMutS for short) and uses thereof.

Background

With the rise of synthetic biology, we have entered the age of creating genes with new gene functions, even whole genomes. DNA synthesis has a wide range of applications, including gene synthesis, synthesis of metabolic pathways, and even synthesis of the entire genome (Gibson et al, 2008. De novo gene synthesis, an important technical approach in synthetic biology, mainly involving the assembly of short-chain oligonucleotides (oligos) into long-chain DNA by PCR-or DNA ligase-based ligation reactions (Jingdong et al, 2004), plays an increasingly important role in the fields of synthetic biology and biotechnology (Bayer and Smolke,2005, carr and church, 2009. At present, oligonucleotides synthesized on porous silica beads are generally used as starting oligonucleotides for synthesis reactions, which are relatively expensive and have low synthesis throughput. Compared with the traditional method, the cost of the oligonucleotide is reduced and the synthesized flux is greatly improved by synthesizing the oligonucleotide on a solid phase plane (microfluidic chip) in parallel by utilizing a microfluidic synthesis technology (Gao et al, 2001, quan et al, 2011 richmond et al, 2004. However, the efficiency of each step of the microchip synthesis reaction is lower than that of the conventional method and has a relatively high error rate, resulting in microchip synthesis with lower synthesis quality and shorter oligonucleotide length (Tian et al, 2009). Errors in the synthesis of genes result from erroneous bases introduced during chemical synthesis of oligonucleotides, extension by polymerase and erroneous assembly between oligonucleotides (Cline et al, 1996). Due to the presence of synthesis errors, it is often unavoidable to use clonal sequencing methods to select a clone with the correct sequence from a plurality of clones, and the cost of this fraction usually accounts for a large fraction of the total cost of gene synthesis. Therefore, improving the fidelity of synthetic DNA is an urgent requirement for large-scale application of gene de novo synthesis technology. Enzymatic error correction methods are cost effective methods of error correction. Typically, these enzymes specifically recognize and bind DNA containing mismatched sequences. Thus, the DNA duplex randomly combines the wrong single strand and the correct single strand by denaturing annealing, allowing the missynthesised bases to form mismatches, and then deleting the synthesis error using a mismatch binding protein (MutS) or a mismatch excising enzyme (e.g., resolvase). Lubock et al systematically compared six different error correcting enzymes and concluded that: mutS is best suited to increase the number of perfect assemblies (up to 25.2-fold) (Lubock et al, 2017).

The mismatch repair system (MMR) system, which is ubiquitous in eukaryotes and prokaryotes, ensures accurate and stable replication of genetic material. The mismatch binding protein MutS is an important component of MMR that recognizes And binds many different DNA mismatches (ad And Jinksrobertson,2000, iayer et al, 2006. Although proteins differ in their ability to bind different mismatches, they can bind almost all single base mismatches as well as 1-4 base insertion or deletion mutations (Whitehouse et al, 1997). MutS from E.coli (EcoMutS) is about 97kDa and is homologous to G: t, A: c, A: a, G: the binding efficiency of G was higher than other mismatches (Brown et al, 2001). MutS asymmetrically binds to mismatched DNA as a homodimer.

MutS has been reported to be useful for correcting gene fragments for oligonucleotide assembly (Binkowski et al, 2005. MutS from Thermus aquaticus (TaqMutS) is commonly used for error correction because it is more thermostable than EcoMutS and has a weak binding ability to fully complementary DNA. However, taqMutS binds to mismatched bases much less strongly than EcoMutS, which directly reduces the error correction efficiency (Brown et al, 2001. Recently, high throughput error correction methods for correcting synthesis errors in chip synthesized oligonucleotide libraries and their assembled genes have been established (Wan et al, 2017, wen et al, 2014. By using the method, the proteins EcoMutS and TaqMutS with mismatch binding capacity can be immobilized on a cellulose column, and the scheme has low cost and high throughput. However, the poor thermal stability of EcoMutS limits the large-scale application of this error correction system in gene synthesis.

Therefore, in view of the above, there is a need in the art for mismatch binding protein mutants with higher thermostability.

Disclosure of Invention

The invention aims to provide a mutant of a mismatch binding protein and application of the mutant.

In a first aspect, the present invention provides a mismatch binding protein that:

a. comprising the amino acid sequence shown as SEQ ID NO 52 and having mutations selected from at least one of the group consisting of:

1) Serine at both position 120 and 151 were mutated to cysteine,

2) Leucine at position 157 and glycine at position 233 were both mutated to cysteine,

3) Glutamic acid at position 451 and valine at position 465 are both mutated to cysteine,

4) Both the methionine at position 609 and the threonine at position 723 are mutated into cysteine, or

5) Serine at position 120, leucine at position 157, glutamic acid at position 451, and methionine at position 609 are all mutated to cysteine; or

b. A mismatch-binding protein derived from a) and having a sequence obtained by substituting, deleting or adding 1 to 20 amino acid residues from the sequence defined in a), and having substantially the function of the mismatch-binding protein defined in a). In a preferred embodiment, the mismatch binding protein is derived from an escherichia bacterium, preferably, escherichia coli.

In a preferred embodiment, the mismatch binding protein:

a. has an amino acid sequence as shown in SEQ ID NO 52 and has at least one mutation selected from the group consisting of:

1) Serine at both position 120 and 151 were mutated to cysteine,

2) Leucine at position 157 and glycine at position 233 were both mutated to cysteine,

3) Glutamic acid at position 451 and valine at position 465 are both mutated to cysteine,

4) Both the methionine at position 609 and the threonine mutation at position 723 are cysteine, or

5) Serine at position 120, leucine at position 157, glutamic acid at position 451, and methionine at position 609 are all mutated to cysteine; or

b. A mismatch binding protein derived from a) having a sequence defined by a) by substitution, deletion or addition of one or several amino acid residues, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 3, most preferably 1 amino acid residue, and having substantially the function of the mismatch binding protein defined by a).

In a preferred embodiment, the mismatch binding protein:

a. the amino acid sequence is shown as SEQ ID NO 54, 56, 58, 60 or 62; or

b. A mismatch binding protein derived from a) comprising a sequence defined in a) by substitution, deletion or addition of one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 amino acid residue, and having substantially the function of the mismatch binding protein defined in a).

In a preferred embodiment, the amino acid sequence of the mismatch binding protein is as shown in SEQ ID NO 54, 56, 58, 60 or 62.

In a preferred embodiment, the mismatch binding protein retains at least 50% of its activity after being stored for 90 days at 4 ℃; preferably, more than 60% -70% activity; more preferably, 70% to 80% or more, most preferably, 90% or more.

In a preferred embodiment, the mismatch binding protein reduces the error rate of the synthetic gene to 2/kb, preferably to 1/kb, most preferably to 0.56/kb using an immobilized column purification (MICC mismatch correction System) (Wen et al, 2014).

The term "corresponding to" as used herein has the meaning commonly understood by a person of ordinary skill in the art. Specifically, "corresponding to" refers to a position in a sequence corresponding to a specified position in the other sequence after aligning the two sequences by homology or sequence identity.

In a second aspect, the present invention provides a gene encoding a protein according to the first aspect of the invention.

In a preferred embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO 53, 55, 57, 59 or 61.

In a third aspect, the present invention provides a vector comprising a gene encoding the second aspect of the invention.

In a fourth aspect, the present invention provides a host cell comprising a gene encoding the gene of the second aspect of the invention.

In a preferred embodiment, the host cell is from the genus Escherichia (Escherichia), corynebacterium (Corynebacterium), brevibacterium (Brevibacterium sp.), bacillus (Bacillus), serratia (Serratia) or Vibrio (Vibrio).

In a preferred embodiment, the host cell is e.

In a preferred embodiment, the host cell has the coding gene of the second aspect of the invention integrated into its chromosome or contains the vector of the third aspect of the invention.

In a preferred embodiment, the host cell expresses the mismatch binding protein.

In a fifth aspect, the present invention provides a method of making a mismatch binding protein, the method comprising the steps of:

a. culturing the host cell of the fourth aspect of the invention to produce the mismatch binding protein; and

b. isolating the mismatched binding protein from the culture medium.

In a preferred embodiment, the method is carried out at a culture temperature of 16-45 deg.C, more preferably at a culture temperature of 16 deg.C.

In a sixth aspect, the present invention provides the use of a mismatch binding protein according to the first aspect of the invention for recognizing and binding to a mismatch DNA.

In a preferred embodiment, the mismatch binding protein according to the first aspect of the present invention is used in a free liquid state for recognizing and binding mismatch DNA or is used in combination with EGFP (enhanced green fluorescent protein), CBM (Cellulose binding module) to immobilize the mismatch binding protein on a column for recognizing and binding mismatch DNA. Preferably, the mismatch binding protein according to the first aspect of the invention is used in combination with EGFP, CBM for immobilisation of the mismatch binding protein on a column with increased thermal stability.

In a seventh aspect, the present invention provides a method of making a mismatch binding protein according to the first aspect of the invention, said method comprising the steps of:

a. modifying the coding sequence of the amino acid sequence shown in SEQ ID NO. 52 so that the amino acid sequence corresponding to the amino acid sequence shown in SEQ ID NO. 52 in the coded amino acid sequence has at least one group of mutations selected from:

1) Serine at both position 120 and 151 were mutated to cysteine,

2) Leucine at position 157 and glycine at position 233 were both mutated to cysteine,

3) Glutamic acid at position 451 and valine at position 465 are both mutated to cysteine,

4) The methionine at position 609 and the threonine at position 723 are both cysteine, or

5) Serine at position 120, leucine at position 157, glutamic acid at position 451, and methionine at position 609 are all mutated to cysteine;

b. transfecting the coding sequence obtained in a) directly into a suitable host cell or introducing the coding sequence into a suitable host cell through a vector;

c. culturing the host cell obtained in b);

d. isolating from the culture system obtained in step c) the mismatched binding protein produced by the host cell; and

e. the enzymatic activity and the thermal stability of the mismatch binding protein are determined.

In an eighth aspect, the present invention provides a method of engineering a wild-type mismatch binding protein to increase thermostability, the method comprising the steps of:

a. comparing the amino acid sequence of the wild type mismatch binding protein with the amino acid sequence shown in SEQ ID NO. 52; and

b. modifying the coding sequence of the wild-type mismatch binding protein such that the amino acid sequence encoded has a mutation corresponding to the amino acid sequence shown in SEQ ID NO. 52 selected from at least one of the following:

1) Serine at both position 120 and 151 were mutated to cysteine,

2) Both the leucine at position 157 and the glycine at position 233 were mutated to cysteine,

3) Glutamic acid at the 451 st position and valine at the 465 th position are both mutated to cysteine,

4) Both the methionine at position 609 and the threonine mutation at position 723 are cysteine, or

5) Serine at position 120, leucine at position 157, glutamic acid at position 451, and methionine at position 609 are all mutated to cysteine;

c. transfecting the coding sequence obtained in the step b directly into a suitable host cell or introducing the coding sequence into a suitable host cell through a vector;

d. culturing the resulting host cell;

e. isolating the mismatched binding protein produced by the host cell from the culture system obtained in step d; and

f. the enzyme activity and the thermal stability of the mismatch binding protein mutant are determined.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a construction diagram of a plasmid of the present invention.

Figure 2 compares the thermal stability of the mutant and wild-type eMutS of the invention at 4 ℃.

A. Results processed with the software "ImageJ".

According to the brightness result of DNA on a DNA agarose gel, utilizing ImageJ software to give a RawIntDen value to all samples, wherein the RawIntDen ratio refers to the RawIntDen value of the sample/the RawIntDen value of a control eMutS, and if the RawIntDen ratio is lower than 0.3, the protein is active; if the RawIntDen ratio is higher than 0.3, the protein is inactivated.

B. Thermostability of different eMutS protein mutants.

eMuts is a wild-type control, eMuts-E38C-G70C, eMuts-S120C-S151C, eMuts-L157C-G233C, eMuts-E177C-G195C, eMuts-Y474C-R500C, eMuts-E451C-V465C, eMuts-T581C-K644C, eMuts-H585C-Q626C, eMuts-I597C-H760C, eMuts-M609C-T723C, and eMuts-S120C-L157C-E451C-M609C are different eMus protein mutant samples.

Detailed Description

The inventors have conducted extensive and intensive studies and unexpectedly found that the mismatch binding protein mutant obtained by performing site-directed mutagenesis on mismatch binding protein derived from Escherichia coli to increase disulfide bonds can maintain more than 90% of activity after being stored at 4 ℃ for 90 days. The present invention has been completed based on this finding.

The application and the advantages of the invention are as follows:

1. the various mismatch binding proteins provided by the invention can efficiently recognize and bind with mismatched DNA;

2. compared with wild type mismatch binding protein which is not modified, the various mismatch binding proteins provided by the invention have higher thermal stability and wide industrial application prospect.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

Reagents and strains: all reagents in the present invention are commercially available reagents of grade or higher. Wherein, tryptone, yeast extract, naCl, plasmid extraction kit, gel recovery kit and all restriction enzymes are all from Shanghai Biotechnology engineering company. PrimeSTAR HS DNA polymerase, solution I ligase, and pMD18-T vector were purchased from Dalibao Bio Inc. Escherichia coli XL10-gold strain as a host strain used for DNA manipulation (Stratagene, calif.) Luria-Bertani (LB) medium containing 100. Mu.g/ml ampicillin was used for E.coli culture. The plasmid pEcoMutS-CBM3-EGFP (Wen et al, 2014) containing the wild-type eUTS gene having the sequence shown in SEQ ID NO. 51 was gifted by professor of the university of Chinese science and technology and was currently stored in the laboratory where the present inventors are present. LB medium (5 g/l yeast extract, 10g/l tryptone, 5g/l NaCl) was used for induction of the expression medium.

Example 1 preparation of strains:

1. construction of various plasmid vectors of the invention:

1) Extracting a plasmid of pEcoMutS-CBM3-EGFP (donated by professor of China scientific and technical university), using the extracted plasmid of pEcoMutS-CBM3-EGFP as a template, carrying out PCR by using KOD Plus Neo DNA polymerase (TOYOBO), primers E38C-G70C-F1 (SEQ ID No. 01) and E38C-G70C-R1 (SEQ ID No. 02) and carrying out enzyme digestion on an amplification product by using Dpn I to obtain a site-mutated plasmid pZJQIBEBT001a. Then, using plasmid pZJQIBEBT001a as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers E38C-G70C-F2 (SEQ ID No. 03) and E38C-G70C-R2 (SEQ ID No. 04) to perform PCR amplification to obtain a pEcoMutS-E38C-G70C-CBM3-EGFP whole plasmid, and using Dpn I to perform enzyme digestion treatment to obtain a plasmid with two site mutations, which is named as pZJQIBEBT001.

The specific operation steps are as follows:

firstly, extracting plasmids, wherein the extraction steps are as follows:

a. 2ml of LB medium containing the corresponding antibiotic were added to a 15ml tube, inoculated with a single colony, closed with a cotton plug and incubated overnight at 37 ℃ with vigorous shaking (250 rpm).

b. 1.5ml of the culture was transferred to a centrifuge tube and centrifuged at 4 ℃ for 30 seconds (12,000 Xg) using a desk top centrifuge.

c. Discard the supernatant, dry the liquid medium in the centrifuge tube with filter paper, suspend the bacterial pellet in 200ml STET buffer, mix well with a vortex mixer.

d. Adding 4 ml of freshly prepared lysozyme solution, mixing, and standing at room temperature for 5min.

e. The centrifuge tube is erected by a float, placed in a boiling water bath, accurately timed for 45 seconds, and immediately centrifuged for 10min after being taken out.

f. The precipitate in the centrifuge tube was picked with a sterile toothpick and discarded, the supernatant of the centrifuge tube was added with 8ml 5% CTAB, and after mixing with a mixer, the mixture was centrifuged at high speed for 5min, the supernatant was discarded, and the liquid in the centrifuge tube was sucked dry with a filter paper.

g. Adding 300 ml of 1.2M sodium chloride, fully dissolving the precipitate, adding 750 ml of cold ethanol, fully mixing, centrifuging for 15min, and removing the supernatant.

h. Taking 1ml of 70% cold ethanol, slowly rinsing the inner wall of the centrifugal tube, and centrifuging for 5min. Discarding the supernatant, sucking the liquid on the tube wall with filter paper, and naturally drying the nucleic acid precipitate at room temperature for 5-10min.

i. The precipitate was dissolved in 50 ml of TE buffer and mixed well in a mixer. Thus obtaining plasmid preparation liquid, and the prepared plasmid is used for the next experiment.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) And (3) amplifying a PCR system of a pEcoMutS-E38C-CBM3-EGFP whole plasmid by taking the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And carrying out enzyme digestion on the amplification product pEcoMutS-E38C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a first point mutation plasmid pZJQIBEBT001a.

(1) An enzyme digestion system of pEcoMutS-E38C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT001a (FIG. 1).

(3) And (3) amplifying a PCR system of a pEcoMutS-E38C-G70C-CBM3-EGFP whole plasmid by taking the pZJQIBEBT001a plasmid as a template:

PCR procedure

(4) And (3) carrying out enzyme digestion on the amplification product pEcoMutS-E38C-G70C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a second point mutation plasmid pZJQIBEBT001.

(1) An enzyme digestion system of pEcoMutS-E38C-G70C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid, which had been subjected to site-directed mutagenesis at the second site, was designated pZJQIBEBT001 (FIG. 1).

2) PCR was performed using KOD Plus Neo DNA polymerase (TOYOBO), primers S120C-S151C-F1 (SEQ ID No. 05) and S120C-S151C-R1 (SEQ ID No. 06) using the extracted pEcoMutS-CBM3-EGFP plasmid as a template, and the amplified product was digested with Dpn I to obtain a site-mutated plasmid pZJQIBEBT002a. Then, using plasmid pZJQIBEBT002a as a template, performing PCR amplification by using KOD Plus Neo DNA polymerase (TOYOBO), primers S120C-S151C-F2 (SEQ ID No. 07) and S120C-S151C-R2 (SEQ ID No. 08) to obtain a pEcoMutS-S120C-S151C-CBM3-EGFP whole plasmid, and performing enzyme digestion treatment by Dpn I to obtain a plasmid with two site mutations, wherein the plasmid is named as pZJQIBEBT002.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) And (3) amplifying a PCR system of a pEcoMutS-S120C-CBM3-EGFP whole plasmid by taking the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And carrying out enzyme digestion on the amplified product pEcoMutS-S120C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain the first point mutation plasmid pZJQIBEBT002a.

(1) The restriction enzyme digestion system of pEcoMutS-S120C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT002a (FIG. 1).

(3) The PCR system for amplifying the pEcoMutS-S120C-S151C-CBM3-EGFP whole plasmid by taking the pZJQIBEBT002a plasmid as a template comprises the following steps:

PCR procedure

(4) The amplified product pEcoMutS-S120C-S151C-CBM3-EGFP full plasmid is subjected to enzyme digestion by using Dpn I, and the product is recovered to obtain a plasmid pZJQIBEBT002 with the second point mutation.

(1) An enzyme digestion system of pEcoMutS-S120C-S151C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid, which had been subjected to site-directed mutagenesis at the second site, was designated pZJQIBEBT002 (FIG. 1).

3) PCR was performed using KOD Plus Neo DNA polymerase (TOYOBO), primers L157C-G233C-F1 (SEQ ID No. 09) and L157C-G233C-R1 (SEQ ID No. 10) using the extracted pEcoMutS-CBM3-EGFP plasmid as a template, and the amplified product was digested with Dpn I to obtain a site-mutated plasmid pZJQIBEBT003a. Then, using plasmid pZJQIBEBT003a as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers L157C-G233C-F2 (SEQ ID No. 11) and L157C-G233C-R2 (SEQ ID No. 12) to perform PCR amplification to obtain a pEcoMutS-L157C-G233C-CBM3-EGFP whole plasmid, and using Dpn I to perform enzyme digestion treatment to obtain a plasmid with two site mutations, wherein the plasmid is named as pZJQIBEBT003.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) And (3) amplifying a PCR system of a pEcoMutS-L157C-CBM3-EGFP whole plasmid by taking the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And carrying out enzyme digestion on the amplification product pEcoMutS-L157C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a first point mutation plasmid pZJQIBEBT003a.

(1) An enzyme digestion system of pEcoMutS-L157C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT003a (FIG. 1).

(3) And (3) amplifying a PCR system of a whole plasmid pEcoMutS-L157C-G233C-CBM3-EGFP by taking the pZJQIBEBT003a plasmid as a template:

PCR procedure

(4) And carrying out enzyme digestion on the amplification product pEcoMutS-L157C-G233C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a second point mutation plasmid pZJQIBEBT003.

(1) An enzyme digestion system of pEcoMutS-L157C-G233C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid, which had been subjected to the second site-directed mutagenesis, was designated pZJQIBEBT003 (FIG. 1).

4) PCR was performed using KOD Plus Neo DNA polymerase (TOYOBO), primers E177C-G195C-F1 (SEQ ID No. 13) and E177C-G195C-R1 (SEQ ID No. 14) using the extracted pEcoMutS-CBM3-EGFP plasmid as a template, and the amplified product was digested with Dpn I to obtain a site-mutated plasmid pZJQIBEBT004a. Then, using plasmid pZJQIBEBT004a as a template, performing PCR amplification by using KOD Plus Neo DNA polymerase (TOYOBO), primers E177C-G195C-F2 (SEQ ID No. 15) and E177C-G195C-R2 (SEQ ID No. 16) to obtain a pEcoMutS-E177C-G195C-CBM3-EGFP whole plasmid, and performing enzyme digestion treatment by Dpn I to obtain a plasmid with two site mutations, wherein the plasmid is named as pZJQIBEBT004.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-E177C-CBM3-EGFP by taking pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And carrying out enzyme digestion on the amplified product pEcoMutS-E177C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain the plasmid pZJQIBEBT004a with the first point mutation.

(1) The restriction enzyme digestion system of pEcoMutS-E177C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT004a (FIG. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-E177C-G195C-CBM3-EGFP by taking the plasmid pZJQIBEBT004a as a template:

PCR procedure

(4) And carrying out enzyme digestion on the amplified product pEcoMutS-E177C-G195C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a plasmid pZJQIBEBT004 with second point mutation.

(1) An enzyme digestion system of pEcoMutS-E177C-G195C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid, which had been subjected to site-directed mutagenesis at the second site, was designated pZJQIBEBT004 (FIG. 1).

5) PCR was performed using KOD Plus Neo DNA polymerase (TOYOBO), primers E451C-V465C-F1 (SEQ ID No. 17) and E451C-V465C-R1 (SEQ ID No. 18) using the extracted pEcoMutS-CBM3-EGFP plasmid as a template, and the amplified product was digested with Dpn I to obtain a site-mutated plasmid pZJQIBEBT005a. Then, using the plasmid pZJQIBEBT005a as a template, performing PCR amplification by using KOD Plus Neo DNA polymerase (TOYOBO), primers E451C-V465C-F2 (SEQ ID No. 19) and E451C-V465C-R2 (SEQ ID No. 20) to obtain a pEcoMutS-E451C-V465C-CBM3-EGFP whole plasmid, and performing enzyme digestion treatment by using Dpn I to obtain a plasmid with two site mutations, wherein the plasmid is named as pZJQIBEBT005.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-E451C-CBM3-EGFP by taking pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And carrying out enzyme digestion on the amplification product pEcoMutS-E451C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a first point mutation plasmid pZJQIBEBT005a.

(1) The restriction enzyme digestion system of pEcoMutS-E451C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT005a (FIG. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-E451C-V465C-CBM3-EGFP by taking the pZJQIBEBT005a plasmid as a template:

PCR procedure

(4) The amplified product pEcoMutS-E451C-V465C-CBM3-EGFP full plasmid is subjected to enzyme digestion by using Dpn I, and the product is recovered to obtain a plasmid pZJQIBEBT005 with the second point mutation.

(1) An enzyme digestion system of pEcoMutS-E451C-V465C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid, which had been subjected to site-directed mutagenesis of the second site, was designated pZJQIBEBT005 (FIG. 1).

6) PCR was performed using KOD Plus Neo DNA polymerase (TOYOBO), primers Y474C-R500C-F1 (SEQ ID No. 21) and Y474C-R500C-R1 (SEQ ID No. 22) using the extracted pEcoMutS-CBM3-EGFP plasmid as a template, and the amplified product was digested with Dpn I to obtain a site-mutated plasmid pZJQIBEBT006a. Then, using plasmid pZJQIBEBT006a as a template, pEcomutS-Y474C-R500C-CBM3-EGFP whole plasmid is amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO), primers Y474C-R500C-F2 (SEQ ID No. 23) and Y474C-R500C-R2 (SEQ ID No. 24), and after the plasmid is subjected to enzyme digestion treatment by Dpn I, a plasmid with two site mutations is obtained and named as pZJQIBEBT006.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-Y474C-CBM3-EGFP by taking pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And carrying out enzyme digestion on the amplified product pEcoMutS-Y474C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain the first point mutation plasmid pZJQIBEBT006a.

(1) An enzyme digestion system of pEcoMutS-Y474C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT006a (FIG. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-Y474C-R500C-CBM3-EGFP by taking the plasmid pZJQIBEBT006a as a template:

PCR procedure

(4) And carrying out enzyme digestion on the amplified product pEcoMutS-Y474C-R500C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a second point mutation plasmid pZJQIBEBT006.

(1) An enzyme digestion system of pEcoMutS-Y474C-R500C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid, which was subjected to the second site-directed mutagenesis, was designated pZJQIBEBT006 (FIG. 1).

7) PCR was performed using KOD Plus Neo DNA polymerase (TOYOBO), primers T581C-K644C-F1 (SEQ ID No. 25) and T581C-K644C-R1 (SEQ ID No. 26) using the extracted pEcoMutS-CBM3-EGFP plasmid as a template, and the amplified product was digested with Dpn I to obtain a site-mutated plasmid pZJQIBEBT007a. Then, using plasmid pZJQIBEBT007a as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers T581C-K644C-F2 (SEQ ID No. 27) and T581C-K644C-R2 (SEQ ID No. 28) to perform PCR amplification to obtain a pEcoMutS-T581C-K644C-CBM3-EGFP whole plasmid, and using Dpn I to perform enzyme digestion treatment to obtain a plasmid with two site mutations, which is named as pZJQIBEBT007.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-T581C-CBM3-EGFP by taking pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And carrying out enzyme digestion on the amplified product pEcoMutS-T581C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain the first point mutation plasmid pZJQIBEBT007a.

(1) The restriction enzyme digestion system of pEcoMutS-T581C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT007a (FIG. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-T581C-K644C-CBM3-EGFP by taking the plasmid pZJQIBEBT007a as a template:

PCR procedure

(4) And carrying out enzyme digestion on the amplified product pEcoMutS-T581C-K644C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a plasmid pZJQIBEBT007 with a second point mutation.

(1) An enzyme digestion system of pEcoMutS-T581C-K644C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid, which had been subjected to the second site-directed mutagenesis, was designated pZJQIBEBT007 (FIG. 1).

8) PCR was carried out using KOD Plus Neo DNA polymerase (TOYOBO), primers H585C-Q626C-F1 (SEQ ID No. 29) and H585C-Q626C-R1 (SEQ ID No. 30) using the extracted pEcoMutS-CBM3-EGFP plasmid as a template, and the amplified product was digested with Dpn I to obtain a site-mutated plasmid pZJQIBEBT008a. Then, using plasmid pZJQIBEBT008a as a template, performing PCR amplification by using KOD Plus Neo DNA polymerase (TOYOBO), primers H585C-Q626C-F2 (SEQ ID No. 31) and H585C-Q626C-R2 (SEQ ID No. 32) to obtain a pEcoMutS-H585C-Q626C-CBM3-EGFP whole plasmid, and performing enzyme digestion treatment by Dpn I to obtain a plasmid with two site mutations, wherein the plasmid is named as pZJQIBEBT008.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-H585C-CBM3-EGFP by taking pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And carrying out enzyme digestion on the amplified product pEcoMutS-H585C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain the first point mutation plasmid pZJQIBEBT008a.

(1) An enzyme digestion system of pEcoMutS-H585C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT008a (FIG. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-H585C-Q626C-CBM3-EGFP by taking the pZJQIBEBT008a plasmid as a template:

PCR procedure

(4) And carrying out enzyme digestion on the amplified product pEcoMutS-H585C-Q626C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a plasmid pZJQIBEBT008 with a second point mutation.

(1) An enzyme digestion system of pEcoMutS-H585C-Q626C-CBM3-EGFP whole plasmid:

(2) the plasmid obtained, which had been subjected to the second site-directed mutagenesis, was designated pZJQIBEBT008 (FIG. 1).

9) PCR was performed using KOD Plus Neo DNA polymerase (TOYOBO), primers I597C-H760C-F1 (SEQ ID No. 33) and I597C-H760C-R1 (SEQ ID No. 34) using the extracted pEcoMutS-CBM3-EGFP plasmid as a template, and the amplification product was digested with Dpn I to obtain a site-mutated plasmid pZQIBEBT 009a. Then, using plasmid pZJQIBEBT009a as a template, pEcoMutS-I597C-H760C-CBM3-EGFP whole plasmid was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO), primers I597C-H760C-F2 (SEQ ID No. 35) and I597C-H760C-R2 (SEQ ID No. 36), and the plasmid was cleaved with Dpn I to obtain a plasmid with two site mutations, which was named pZJQIBEBT009.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) And (3) amplifying a PCR system of a whole plasmid pEcoMutS-I597C-CBM3-EGFP by taking pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And (3) carrying out enzyme digestion on the amplification product pEcoMutS-I597C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a first point mutation plasmid pZJQIBEBT009a.

(1) An enzyme digestion system of pEcoMutS-I597C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT009a (FIG. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-I597C-H760C-CBM3-EGFP by taking pZJQIBEBT009a plasmid as a template:

PCR procedure

(4) And carrying out enzyme digestion on the amplification product pEcoMutS-I597C-H760C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a second point mutation plasmid pZJQIBEBT009.

(1) An enzyme digestion system of pEcoMutS-I597C-H760C-CBM3-EGFP whole plasmid:

(2) the plasmid obtained after the second site-directed mutagenesis was designated pZJQIBEBT009 (FIG. 1).

10 Using the extracted pEcoMutS-CBM3-EGFP plasmid as a template, carrying out PCR by using KOD Plus Neo DNA polymerase (TOYOBO), primers M609C-T723C-F1 (SEQ ID No. 37) and M609C-T723C-R1 (SEQ ID No. 38), and carrying out enzyme digestion treatment on an amplification product by using Dpn I to obtain a plasmid pZJQIBEBT010a with one site mutation. Then, using plasmid pZJQIBEBT010a as a template, carrying out PCR amplification by using KOD Plus Neo DNA polymerase (TOYOBO), primers M609C-T723C-F2 (SEQ ID No. 39) and M609C-T723C-R2 (SEQ ID No. 40) to obtain a pEcoMutS-M609C-T723C-CBM3-EGFP whole plasmid, carrying out enzyme digestion treatment by Dpn I to obtain a plasmid with two site mutations, and naming the plasmid as pZJQIBEBT010.

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-M609C-CBM3-EGFP by taking pEcoMutS-CBM3-EGFP plasmid as a template:

PCR procedure

(2) And carrying out enzyme digestion on the amplification product pEcoMutS-M609C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain the first point mutation plasmid pZJQIBEBT010a.

(1) An enzyme digestion system of pEcoMutS-M609C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid subjected to site-directed mutagenesis was designated pZJQIBEBT010a (FIG. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-M609C-T723C-CBM3-EGFP by taking the plasmid pZJQIBEBT010a as a template:

PCR procedure

(4) And carrying out enzyme digestion on the amplified product pEcoMutS-M609C-T723C-CBM3-EGFP whole plasmid by using Dpn I, and recovering the product to obtain a plasmid pZJQIBEBT010 with a second point mutation.

(1) The restriction enzyme digestion system of pEcoMutS-M609C-T723C-CBM3-EGFP whole plasmid:

(2) the obtained plasmid, which had been subjected to site-directed mutagenesis at the second site, was designated pZJQIBEBT010 (FIG. 1).

10 Using KOD Plus Neo DNA polymerase (TOYOBO), primers eUTS-NheI-F (SEQ ID No. 41) and eUTS-340-R (SEQ ID No. 42), eUTS-310-F (SEQ ID No. 43) and eUTS-120C-460-R (SEQ ID No. 44) as templates, respectively, with the extracted pEcomutS-CBM3-EGFP plasmid, pZJQIBEBT002 plasmid, pZJQIBEBT003 plasmid, pZJQIBEBT006 plasmid, pZJQIBEBT010 plasmid, eUTS-430-F (SEQ ID No. 45) and eUTS-800-R (SEQ ID No. 46), eUTS-770-F (SEQ ID No. 47) and eUTS-1500-R (SEQ ID No. 48), eUTS-1470-F (SEQ ID No. 49) and eUTS-SacI-R (SEQ ID No. 50) were subjected to PCR to obtain products eUTS-1-340, S120C-310-460, L157C-430-800, E451C-770-1500, and M609C-1470-terminator. Then, the amplified product was subjected to fusion PCR using KOD Plus Neo DNA polymerase (TOYOBO), primers eUTS-NheI-F (SEQ ID No. 41) and eUTS-SacI-R (SEQ ID No. 50) to obtain a product eUTS-S120C-L157C-E451C-M609C. The fragment eMutS-S120C-L157C-E451C-M609C and the plasmid pEcoMutS-CBM3-EGFP are respectively digested by NheI and SacI, connected and transformed to obtain a plasmid with eight site mutations, which is named as pZJQIBEBT011 (figure 1).

The specific operation steps for obtaining the mutant plasmid are as follows:

(1) The PCR system for amplifying the eUTS-1-340 fragment by taking pEcoMutS-CBM3-EGFP plasmid as a template and eUTS-NheI-F (SEQ ID No. 41) and eUTS-340-R (SEQ ID No. 42) as primers:

PCR procedure

(2) A PCR system for amplifying eUTS-1-340 segments by taking pZJQIBEBT002 plasmid as a template and eUTS-310-F (SEQ ID No. 43) and eUTS-S120C-460-R (SEQ ID No. 44) as primers:

PCR procedure

(3) A PCR system for amplifying the L157C-430-800 fragment by taking pZJQIBEBT003 plasmid as a template and eUTS-430-F (SEQ ID No. 45) and eUTS-800-R (SEQ ID No. 46) as primers:

PCR procedure

(4) A PCR system for amplifying the E451C-770-1500 fragment by taking pZJQIBEBT006 plasmid as a template and eUTS-770-F (SEQ ID No. 47) and eUTS-1500-R (SEQ ID No. 48) as primers:

PCR procedure

(5) A PCR system for amplifying an M609C-1470-tail fragment by taking pZJQIBEBT010 plasmid as a template and eUTS-1470-F (SEQ ID No. 49) and eUTS-SacI-R (SEQ ID No. 50) as primers:

PCR procedure

(6) Then performing fusion PCR on the amplified product by using KOD Plus Neo DNA polymerase (TOYOBO), primers eUTS-NheI-F (SEQ ID No. 41) and eUTS-SacI-R (SEQ ID No. 50) to obtain a PCR system of a product eUTS-S120C-L157C-E451C-M609C:

PCR procedure

(7) The fragment eUTS-S120C-L157C-E451C-M609C and the plasmid pEcoMutS-CBM3-EGFP are respectively digested by NheI and SacI, are connected, and are transformed to obtain a plasmid with eight site mutations, namely pZJQIBEBT011 (figure 1).

(1) An enzyme digestion system of a fragment eUTS-S120C-L157C-E451C-M609C:

(2) the restriction system of the plasmid pEcoMutS-CBM 3-EGFP:

(3) the cleavage products were ligated by using Solution I (TAKATA) to obtain a plasmid containing 8 site-directed point mutations, which was designated pZJQIBEBT011 (FIG. 1).

2. Transferring the constructed vector into an expression vector escherichia coli BL 21:

preparation of E.coli BL21 as a host strain into which 10 plasmids subjected to site-directed mutagenesis were transformed

1) And E, chemical transformation of Escherichia coli:

(1) taking out a tube (100 ul) of allelochemicals from a-70 ℃ ultra-low temperature freezer, immediately heating and melting by fingers, inserting the tube on ice, and carrying out ice bath for 5-10min. .

(2) Add 5ul of ligated plasmid mixture (DNA content not more than 100 ng), gently shake and place on ice for 20min.

(3) After being shaken lightly, the mixture is inserted into a water bath with the temperature of 42 ℃ for 45s for heat shock, and then is quickly put back into ice and stands for 3 to 5min.

(4) 500ul of LB medium (containing no antibiotics) was added to each tube in a clean bench, mixed gently, and then fixed on a spring holder of a shaker for 1h at 37 ℃.

(5) Taking 100-300ul of the converted mixed solution from a clean bench, dripping the mixed solution into solid LB plate culture dishes containing suitable antibiotics respectively, and uniformly coating the mixed solution by using a glass coating rod burnt by an alcohol burner.

(6) Marking on the coated culture dish, placing in a constant temperature incubator at 37 ℃ for 30-60 min until all the liquid on the surface permeates into the culture medium, then inverting and placing in the constant temperature incubator at 37 ℃ overnight.

(7) The colonies on the plate were picked and cultured in liquid LB, plasmids were extracted, and the transformation results were identified by PCR.

2) The specific process for constructing various eUTS protein mutant expression strains comprises the following steps:

the plasmid pZJQIBEBT001 containing the mutations at E38C and G70C was transformed into BL21 E.coli. The strain is restored to have the function of Amp resistance, and the function of eMuts is newly added. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT001;

the plasmid pZJQIBEBT002 containing the mutations at the S120C-position and the S151C-position was transformed into BL21 E.coli. The strain will be restored to the Amp resistance function and the function of eMuts will be increased. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT002;

the plasmid pZJQIBEBT003 containing the mutations at the C positions L157 and G233 was transformed into BL21 E.coli. The strain is restored to have the function of Amp resistance, and the function of eMuts is newly added. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT003;

the plasmid pZJQIBEBT004 containing the mutations at E177C-position and G195C-position was transformed into BL21 E.coli. The strain is restored to have the function of Amp resistance, and the function of eMuts is newly added. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT004;

the plasmid pZJQIBEBT005, which contained the E451C-and V465C-mutations, was transformed into BL21 E.coli. The strain is restored to have the function of Amp resistance, and the function of eMuts is newly added. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT005;

the plasmid pZJQIBEBT006 containing the mutations at the C positions Y474 and R500 was transformed into BL21 E.coli. The strain will be restored to the Amp resistance function and the function of eMuts will be increased. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT006;

the plasmid pZJQIBEBT007 containing the mutations at T581C-site and K644C-site was transformed into BL21 E.coli. The strain is restored to have the function of Amp resistance, and the function of eMuts is newly added. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT007;

the plasmid pZJQIBEBT008, which contains the mutations at the C-positions H585 and Q626, was transformed into BL21 E.coli. The strain is restored to have the function of Amp resistance, and the function of eMuts is newly added. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT008;

plasmid pZJQIBEBT009, which contained mutations at I597C and H760C, was transformed into BL21 E.coli. The strain is restored to have the function of Amp resistance, and the function of eMuts is newly added. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT009;

the plasmid pZJQIBEBT010, which contains the M609C-and T723C-mutations, was transformed into BL21 E.coli. The strain is restored to have the function of Amp resistance, and the function of eMuts is newly added. Positive clones were selected on an Amp-resistant medium (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l) and the resulting transformed strain was named: EZJQIBEBT010;

3) Plasmids were extracted and positive strains transformed by E.coli were identified by PCR.

The method comprises the steps of escherichia coli plasmid extraction:

(1) harvesting bacteria: the overnight-cultured (37 ℃ C., 12-16 hours) bacterial solution was centrifuged at room temperature ≧ 10,000g for 1-2 minutes, and the supernatant was discarded completely.

(2) Resuspending: 250 μ l of RNase A-containing cell suspension (S1) was added and the suspension was made sufficient or the bacteria were dispersed thoroughly by repeated pipetting with a pipette tip.

(3) Cracking: add 250. Mu.l of cell lysis solution (S2), mix gently upside down for 5 times, stand at room temperature for 1-5 minutes until the bacteria are lysed sufficiently and the solution becomes translucent.

(4) Neutralization: add 350. Mu.l of neutralization buffer (S3), mix 5 times by gently inverting up and down, mix well, avoid violent shaking. Centrifugation is carried out for 10 minutes at room temperature under the condition of ≧ 12,000g.

(5) DNA binding: carefully sucking the supernatant, transferring the supernatant into a centrifugal adsorption column inserted into the collection tube, centrifuging for 1 minute at room temperature of ≧ 12,000g, discarding the waste liquid in the collection tube, and reinserting the centrifugal adsorption column into the collection tube.

(6) Cleaning: add 500. Mu.l of rinsing solution (WB, please confirm that ethanol is added!) to the column, centrifuge at room temperature ≧ 12,000g for 30 seconds, discard the waste from the collection tube, and reinsert the column back into the collection tube.

(7) Cleaning: add 500. Mu.l of rinsing solution (WB, please confirm that ethanol is added!) to the column, centrifuge at room temperature ≧ 12,000g for 30 seconds, discard the waste from the collection tube, and reinsert the column back into the collection tube. The centrifugation and adsorption column was uncapped and centrifuged again for 2 minutes to completely remove the residual rinse. .

(8) Elution: the column was carefully removed and placed in a new 1.5ml sterile centrifuge tube. 100. Mu.l of Elution Buffer (EB) was added to the center of the silica gel adsorption membrane, and after standing at room temperature for 1 minute, plasmid DNA was collected by centrifugation at ≧ 12,000g for 1 minute.

(9) Storing: the centrifugal adsorption column is discarded, and the purified plasmid can be directly used for subsequent reaction or stored at the temperature of minus 20 ℃ for a long time.

Identifying a PCR system of positive strains transformed by escherichia coli:

(1) PCR system containing plasmid of eMutS:

PCR procedure

After PCR amplification, the universal primer M13 is used as a primer and plasmid is used as a template, a strain with a gene band can be specifically amplified, namely a positive strain, and the next experiment is carried out. The size of the gene is 2586bp.

Example 2 thermostability of mutant eMuts proteins

This example was used to compare the level of protease activity stability expressed by strains of different eMutS mutants. The result shows that the protein eMuts-S120C-L157C-E451C-M609C expressed by the EZJQIBEBT011 strain has the highest activity stability.

1. The strains were recovered on LB medium plates. Control strain: eMutS. Experimental strains: eUTS-E38C-G70C, eUTS-Y474C-R500C, eUTS-T581C-K644C, eUTS-I597C-H760C, eUTS-S120C-S151C, eUTS-L157C-G233C, eUTS-E451C-V465C, eUTS-M609C-T723C, eUTS-E177C-G195C, eUTS-H585C-Q626C, and eUTS-S120C-L157C-E451C-M609C. Incubated at 37 ℃ for 1 day.

2. Single clones were picked up separately and inoculated in 5ml of liquid LB medium. 37 ℃,250rpm, overnight.

3. A36-liter conical flask was prepared and filled with 100mL of YPD medium. The formula is as follows: 10g/l of tryptone, 5g/l of yeast extract and 10g/l of NaCl. And (5) sterilizing for later use.

4. An appropriate amount of the overnight culture was inoculated into 200ml of LB medium at an inoculation ratio of 1, 1000,37 ℃ and 250rpm for culture.

And 5.OD600=0.8, collecting bacteria, crushing bacteria, purifying protein, and measuring enzyme activity.

6. As can be seen from FIG. 2, the mutants eUTS-E38C-G70C and eUTS-Y474C-R500C lose enzyme activity. The enzyme activities of the mutant eUTS-T581C-K644C and eUTS-I597C-H760C can last for 21 days, which is similar to that of a control group. The enzyme activity duration of the mutants eMuts-S120C-S151C, eMuts-L157C-G233C, eMuts-E451C-V465C and eMuts-M609C-T723C, and eMuts-S120C-L157C-E451C-M609C is longer than that of the control group, and the enzyme activity of the mutants eMuts-S120C-L157C-E451C-M609C can last for 90 days.

Example 3 error correction capability of eMuts protein mutants to bind mismatched DNA

This example was used to verify the error correcting ability of the proteases expressed by two strains of eMutS mutants with the strongest enzyme activity stability. The results show that the error correcting ability of the protease with improved stability is still maintained. Among them, the error correction effect of the eUTS-S120C-L157C-E451C-M609C is the best, even better than that of the strain before mutation.

1. The strains were recovered on LB medium plates. Control strain: eUTS. Experimental strains: eUTS-S120C-S151C, eUTS-L157C-G233C, eUTS-S120C-L157C-E451C-M609C. Incubated at 37 ℃ for 1 day.

2. Single clones were picked up separately and inoculated in 5ml of liquid LB medium. 37 ℃,250rpm, overnight.

3. 100mL of 12 bottles of YPD medium were prepared and distributed in 1L conical flasks. The formula is as follows: 10g/l of tryptone, 5g/l of yeast extract and 10g/l of NaCl. And (5) sterilizing for later use.

4. An appropriate amount of the overnight culture was inoculated into 200ml of LB medium at an inoculation ratio of 1, 1000,37 ℃ and 250rpm for culture.

And 5.OD600=0.8, collecting bacteria, crushing bacteria, purifying protein, and using the protein in DNA error correction experiment.

6. All eMutS proteins were used with the MICC system in application experiments, which were reported in past studies. The correction system with improved stability is used for correcting the XR and Cas9 homologous genes synthesized by MCP-oligos. As shown in Table 1, the error rate of the oligomer was about 1.2%. Debugging errors using MICC significantly reduced the error rate in synthesizing XR and Cas9 homologous genes. Experiments show that the eMuts protein stability is improved by the disulfide bond construction, and the original error correction capability is kept. Among the strains, the eMuts-S120C-L157C-E451C-M609C not only greatly improves the thermal stability, but also has the best error correction effect, and is even better than the strains before mutation.

TABLE 1 results of DNA error correction of eMuts mutants

It should be understood that while the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein, and any combination of the various embodiments may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Sequence listing

<110> institute of bioenergy and Process in Qingdao, china academy of sciences

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<211> 34

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

gttcacgctc gcggacgcac agacgctcca gata 34

<210> 19

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

ctggacacgc tgaaatgcgg ctttaatgcg gtg 33

<210> 20

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

caccgcatta aagccgcatt tcagcgtgtc cag 33

<210> 21

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

gcggtgcacg gctactgcat tcaaatcagc cgt 33

<210> 22

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

acggctgatt tgaatgcagt agccgtgcac cgc 33

<210> 23

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

ctgaaaaacg ccgagtgcta catcattcca gag 33

<210> 24

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

ctctggaatg atgtagcact cggcgttttt cag 33

<210> 25

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

ccgggcattc gcatttgcga aggtcgccat ccg 33

<210> 26

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

cggatggcga ccttcgcaaa tgcgaatgcc cgg 33

<210> 27

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

tatgtaccgg cacaatgcgt cgagattgga cct 33

<210> 28

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

aggtccaatc tcgacgcatt gtgccggtac ata 33

<210> 29

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

attaccgaag gtcgctgccc ggtagttgaa caa 33

<210> 30

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

ttgttcaact accgggcagc gaccttcggt aat 33

<210> 31

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

agtacctata tgcgctgcac cgcactgatt gcg 33

<210> 32

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

cgcaatcagt gcggtgcagc gcatataggt act 33

<210> 33

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

ctgaatgagc cattttgcgc caacccgctg aat 33

<210> 34

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

attcagcggg ttggcgcaaa atggctcatt cag 33

<210> 35

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

accattgcct ttatgtgcag cgtgcaggat ggc 33

<210> 36

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

gccatcctgc acgctgcaca taaaggcaat ggt 33

<210> 37

<211> 34

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

tcgccgcagc gccgctgctt gatcatcacc ggtc 34

<210> 38

<211> 34

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

gaccggtgat gatcaagcag cggcgctgcg gcga 34

<210> 39

<211> 36

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

taagattaag gcattgtgct tatttgctac ccacta 36

<210> 40

<211> 36

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

tagtgggtag caaataagca caatgcctta atctta 36

<210> 41

<211> 31

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

ctagctagca tgagtgcaat agaaaatttc g 31

<210> 42

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

cgatacgcac aactttgcgc 20

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

gtccggttga gcgcaaagtt 20

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

gcccggagca gatatccagc 20

<210> 45

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

gctacgcgac gctggatatc 20

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

atgctgtcct gctcacgttc 20

<210> 47

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

catcaccatg gaacgtgagc 20

<210> 48

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

gcgctcggcg tttttcagcg 20

<210> 49

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

cgtcgccaga cgctgaaaaa cg 22

<210> 50

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

cgagctccac caggctcttc aagcg 25

<210> 51

<211> 2562

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 51

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcgggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatcagc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc agttccgggc gttttcgcct gagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgcggac tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg gaagtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aagttggctt taatgcggtg cacggctact acattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccatttat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgcatgttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgacgt tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggattta tcgcttgaag agcctggtgt aa 2562

<210> 52

<211> 853

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 52

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

Ala Lys Leu Val Asn Gln Gly Glu Ser Val Ala Ile Cys Glu Gln Ile

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Ser Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Ser Ser Gly Arg Phe Arg Leu Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Gly Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Glu Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Val Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Met Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Thr Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 53

<211> 2562

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 53

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcgggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgcct gagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgcggac tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg gaagtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aagttggctt taatgcggtg cacggctact acattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccatttat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgcatgttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgacgt tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggattta tcgcttgaag agcctggtgt aa 2562

<210> 54

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 54

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

Ala Lys Leu Val Asn Gln Gly Glu Ser Val Ala Ile Cys Glu Gln Ile

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Cys Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Cys Ser Gly Arg Phe Arg Leu Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Gly Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Glu Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Val Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Met Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Thr Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 55

<211> 2562

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 55

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcgggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgctg cagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgctgcc tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg gaagtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aagttggctt taatgcggtg cacggctact acattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccatttat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgcatgttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgacgt tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggattta tcgcttgaag agcctggtgt aa 2562

<210> 56

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 56

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

Ala Lys Leu Val Asn Gln Gly Glu Ser Val Ala Ile Cys Glu Gln Ile

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Ser Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Ser Ser Gly Arg Phe Arg Cys Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Cys Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Glu Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Val Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Met Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Thr Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 57

<211> 2562

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 57

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcgggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgcct gagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgcggac tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg tgcgtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aatgcggctt taatgcggtg cacggctact acattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccatttat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgcatgttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgacgt tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggattta tcgcttgaag agcctggtgt aa 2562

<210> 58

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 58

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

Ala Lys Leu Val Asn Gln Gly Glu Ser Val Ala Ile Cys Glu Gln Ile

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Ser Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Ser Ser Gly Arg Phe Arg Leu Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Gly Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Cys Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Cys Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Met Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Thr Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 59

<211> 2562

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 59

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcgggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgcct gagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgcggac tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg gaagtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aagttggctt taatgcggtg cacggctact acattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccatttat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgctgcttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgtgct tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggattta tcgcttgaag agcctggtgt aa 2562

<210> 60

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 60

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

Ala Lys Leu Val Asn Gln Gly Glu Ser Val Ala Ile Cys Glu Gln Ile

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Ser Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Ser Ser Gly Arg Phe Arg Leu Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Gly Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Glu Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Val Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Cys Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Cys Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 61

<211> 2562

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 61

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcgggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgctg cagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgctgcc tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg tgcgtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aatgcggctt taatgcggtg cacggctact acattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccatttat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgctgcttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgtgct tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggattta tcgcttgaag agcctggtgt aa 2562

<210> 62

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 62

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

Ala Lys Leu Val Asn Gln Gly Glu Ser Val Ala Ile Cys Glu Gln Ile

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Cys Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Cys Ser Gly Arg Phe Arg Cys Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Cys Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Cys Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Cys Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Cys Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Cys Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

Claims (11)

1. A mismatch binding protein, wherein the amino acid sequence of said mismatch binding protein is selected from the group consisting of seq id no:

1) 52, and the amino acid sequence is obtained by mutating serine at the 120 th site and serine at the 151 th site of the amino acid sequence shown in SEQ ID NO. 52 into cysteine;

2) 52, wherein both the 157 th leucine and the 233 th glycine of the amino acid sequence are mutated into cysteine to obtain the amino acid sequence; or

3) 52, serine at the 120 th site, leucine at the 157 th site, glutamic acid at the 451 th site and methionine at the 609 th site are mutated into cysteine, and the obtained amino acid sequence is obtained.

2. The mismatch binding protein of claim 1, wherein said mismatch binding protein has an amino acid sequence as set forth in SEQ ID No. 54, SEQ ID No. 56, or SEQ ID No. 62.

3. A gene encoding the mismatch binding protein of claim 1.

4. The encoding gene of claim 3, wherein the sequence of the gene is shown in SEQ ID NO 53, SEQ ID NO 55 or SEQ ID NO 61.

5. A vector comprising the coding gene of claim 3.

6. A host cell comprising the coding gene of claim 3.

7. Use of a host cell according to claim 6 for the production of a mismatch binding protein.

8. A method of producing a mismatch binding protein, said method comprising the steps of:

a. culturing the host cell of claim 6 to produce the mismatch binding protein; and

b. isolating the mismatched binding protein from the culture medium.

9. Use of the mismatch binding protein of claim 1 for recognizing and binding DNA mismatches.

10. The use of claim 9, wherein the mismatch binding protein is used in free liquid form for recognition and binding of mismatch DNA, or in combination with EGFP, CBM to immobilize the mismatch binding protein on a column for recognition and binding of mismatch DNA.

11. A method of engineering a wild-type mismatch binding protein for improved thermostability, the method comprising the steps of:

a. comparing the amino acid sequence of the wild type mismatch binding protein with the amino acid sequence shown in SEQ ID NO. 52;

b. modifying the coding sequence of said wild type mismatch binding protein such that the mutation in the encoded amino acid sequence corresponding to the amino acid sequence as depicted in SEQ ID NO:52 is selected from one of the group consisting of:

1) Serine at both position 120 and 151 were mutated to cysteine,

2) Both the leucine at position 157 and the glycine at position 233 were mutated to cysteine, or

3) Serine at position 120, leucine at position 157, glutamic acid at position 451, and methionine at position 609 are all mutated to cysteine;

c. transfecting the coding sequence obtained from the step b directly into a suitable host cell or introducing the coding sequence into a suitable host cell through a vector;

d. culturing the resulting host cell;

e. isolating from the culture system of step d the mismatch binding protein produced by said host cell; and

f. determining the thermostability of the mismatch binding protein.

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