CN102517241B - Preparation method of mutant of double-carbonyl reductase containing D-amino acid - Google Patents

Abstract

The invention discloses a preparation method of protein and polypeptide containing D-lysine. According to the invention, an expression gene of a target protein is introduced into a specific enzyme-digested multiple cloning site of an expression plasmid, and a target site of the expression gene is mutated into a TAG codon; a sequence of ph aminoacyl tRNA synthetase is introduced into another specific enzyme-digested multiple cloning site of the expression plasmid, such that a recombinant plasmid is obtained; a pAC-ph delta-AK3 plasmid is prepared; the pAC-ph delta-AK3 plasmid and the recombinant plasmid are cotransfected into a host, such that an expression system is obtained; D-lysine is added into a cultivation medium, and is used for inducing the expression system to express a target protein; the protein is purified, such that the target protein is obtained. According to the invention, a characteristic that lysyl tRNA synthetase and inhibitory type tRNA molecule pair originated from pyrococcushorikoshii can specifically select amber codon TAG is adopted, and D-lysine is introduced in a set position in double-carbonyl reductase. The introduction rate is close to 100%. The method has wide application values.

Description

A kind of preparation method of the mutant that contains the amino acid whose di-carbonyl reduction enzyme of D type

Technical field

The invention belongs to biological technical field, relate to a kind of method that contains the amino acid whose protein of D type and/or polypeptide for preparing.

Background technology

Introducing alpha-non-natural amino acid in proteins and peptides, to make it obtain new character be a kind of strategy that has been widely used in protein and peptide class drug modification and has prepared the field such as biomaterial.The method that can in protein and peptide, introduce now alpha-non-natural amino acid mainly comprises 3 kinds:

(1) polypeptide solid-state reaction method.The Solid-phase Polypeptide stepwise synthesis method that at first Merrifield adopts D type amino acid and arginine analog are incorporated in luliberin (referring to: Science. 1965; 150:178-85.).The method can be introduced alpha-non-natural amino acid in optional position in the polypeptide that is less than 100 residues.But have, only limit to synthetic be less than the small peptide of 100 amino-acid residues and need loaded down with trivial details protection and go the shortcomings such as step such as protection.

(2) external protein translation.Larisa M etc. utilizes the external protein translation system of S30 successfully to introduce D-Met and D-Phe(referring to J. Am. Chem. Soc. 2003 in Tetrahydrofolate dehydrogenase and luciferase; 125:6616-7.).But the method has the shortcomings such as efficiency is low, cost is high and can not amplify.

(3) genetic code extending method.The people such as Schultz are by tRNA and the aminoacyl-tRNA synthetase molecule pair of orthogenesis external source in viable cell, the ribosomal protein synthesis system by a series of L-type alpha-non-natural amino acid by viable cell be incorporated in protein (referring to: Science. 2001; 292:498-500.).Obtain a series of protein with new property by this alpha-non-natural amino acid introducing technology and be widely used in association area, as: 1. the band Fluorescent amino acid introduced for associated protein location in cell in albumen; 2. introduce the research for click chemistry with the alpha-non-natural amino acid of ketone groups; 3. introduce the pointed decoration of the alpha-non-natural amino acid of ketone group containing and azido group for the protein and peptide medicine.4. introduce the alpha-non-natural amino acids such as band acetyl, methyl for acetylize in analog cell and the posttranslational modification such as methylate.

Although the genetic code extended technology can fix a point to introduce more than 100 kind of L-type alpha-non-natural amino acid in the albumen of arbitrary size, the technology of comparing forefathers has great breakthrough, but this technology still can not meet the requirement that people wish to come by changing amino acid chiral pointed decoration and engineered protein and polypeptide.Therefore, develop and a kind ofly effectively in the albumen of arbitrary size, introduce pathology that the amino acid whose method of D type is pointed decoration proteins and peptides class medicine, research geriatric disease, prepare the fields such as space structure that novel biomaterial and pointed decoration change protein and all be significant.

Summary of the invention

Goal of the invention of the present invention is to provide a kind of escherichia coli prokaryotic expression system, utilize this escherichia coli prokaryotic expression system to introduce D type Methionin in any site of di-carbonyl reduction enzyme, the polypeptide and/or the protein that contain D type Methionin for preparation provide new method.

Principle of the present invention is: by express a pair of deriving from Bacillus coli cells pyrococcus horikoshiilysyl tRNA synthetic enzyme and the molecule pair of inhibition type tRNA, by paraxin suppress experiment and the methods such as proteolysis and Chiral HPLC analysis proved this molecule to containing in D type Methionin substratum to the preference of D type Methionin, and utilize this Preference to introduce D type Methionin in the nonactive site of di-carbonyl reduction enzyme fixed point, introduce efficiency and approach 100%.

To achieve the above object of the invention, the technical solution used in the present invention is: a kind of escherichia coli prokaryotic expression system, described escherichia coli prokaryotic expression system contains pAC-ph △ with the p15A replicon-AK3 plasmid and with the pETDuet-T2 of ColE1 replicon ^dthe K-DKR plasmid.

In technique scheme, pAC-ph △-AK3 plasmid is obtained by the pACYC184 transformation: 2920 of pACYC184 replace to 3542 bit sequences phthe sequence of aminoacyl-tRNA synthetase, 1425 replace to 3 inhibition tRNA sequences to 1524 bit sequences.PAC-ph △-AK3 can encode and derive from pyrococcus horikoshiilysyl tRNA synthetic enzyme and inhibition type tRNA molecule pair, wherein lysyl tRNA synthetic enzyme is by glutamine-tRNA synthetase promotor and terminator regulating and expressing, inhibition type tRNA is by lpp promotor and rrnC terminator regulating and expressing; In addition, 112 of E.C. 2.3.1.28 aspartic acid codon mutations become amber codon TAG to detect pyrococcus horikoshiilysyl tRNA synthetic enzyme and the right expression of tRNA molecule.Particularly, pAC-ph △-AK3 plasmid has the described nucleotide sequence of SEQ ID No.1.

In technique scheme, pETDuet-T2 ^dk-DKR is obtained by plasmid pETDuet-1 transformation: introduced the sequence of Diketoreductase mutant in first multiple clone site, and 106 to 143 sequences that replace to Diketoreductase mutant, second multiple clone site introduced phthe sequence of aminoacyl-tRNA synthetase, replace and become to 354 bit sequences for 298 phthe sequence of aminoacyl-tRNA synthetase.PETDuet-T2 ^dfirst multiple clone site of K-DKR is connected with the DKR gene of the second bit strip amber mutation; Second multiple clone site is connected with pyrococcus horikoshiilysyl tRNA synthetic enzyme ( phtRNARS) gene; Two fragment genes are all started by T7promoter and are subject to the lactose operon regulating and expressing.Preferably, described pETDuet-T2 ^dthe K-DKR plasmid comprises following four characteristics: second codon of gene of the di-carbonyl reduction enzyme of (1) coding becomes amber codon TAG by the ACC sudden change, (2) N of di-carbonyl reduction enzyme mutant gene end has added the label of His6, (3) mutator gene of di-carbonyl reduction enzyme is building up to first multiple clone site of pETDuet-1, and under T7promoter starts by the lactose operon regulating and expressing.(4) second of pETDuet-1 multiple clone site added phthe gene of tRNARS strengthens phthe expression of tRNARS is to improve the right suppression efficiency of lysyl tRNA synthetic enzyme molecule.Particularly, pETDuet-T2 ^dthe K-DKR plasmid has the described nucleotide sequence of SEQ ID No.2.

PACYC184 and pETDuet-1 are commercial carriers, the U.S. Patent Application Publication specification sheets that the sequence of tRNA and tRNA synthetic enzyme can be US 2006/0177900 referring to publication number, and particularly, the sequence of tRNA refers to shown in SEQ ID No.3; The sequence of tRNA synthetic enzyme refers to shown in SEQ ID No.4.

In technique scheme, described intestinal bacteria are preferably e. coli bl21 (DE3).

In technique scheme, described escherichia coli prokaryotic expression system is by pAC-ph △-AK3 plasmid and pETDuet-T2 ^dk-DKR plasmid co-transfection e. coli bl21 (DE3) cell obtains.

The method of the simultaneously claimed Diketoreductase mutant that utilizes above-mentioned escherichia coli prokaryotic expression system preparation to contain D type Methionin of the present invention comprises the following steps:

(1) add D type Methionin in substratum, isopropylthiogalactoside (IPTG) induces above-mentioned escherichia coli prokaryotic expression system to express, high pressure fragmentation, centrifuging and taking supernatant;

(2) adopt the method purifying supernatant liquor of affinity chromatography and anion-exchange chromatography combination to obtain containing the amino acid whose di-carbonyl reduction enzyme of D type.

The method of the above-mentioned Diketoreductase mutant that utilizes the escherichia coli prokaryotic expression system preparation to contain D type Methionin specifically comprises the following steps:

(1) by pAC-ph △-AK3 plasmid and pETDuet-T2 ^dk-DKR plasmid co-transfection e. coli bl21 (DE3) cell, flat board is coated containing dull and stereotyped upper 37 ℃ of the LB of the paraxin of the penbritin of 4 μ g/ml tsiklomitsins, 40 μ g/ml, 20 μ g/ml and is cultivated 18～20 hours, and on the picking flat board, single bacterium colony is cultivated approximately 20 hours in LB liquid nutrient medium 37 degree containing tsiklomitsin, penbritin, paraxin; Nutrient solution is transferred and made its final OD be about 0.1 in the M9 liquid nutrient medium containing tsiklomitsin, penbritin, paraxin, then 37 degree are cultivated 30 hours, transfer and make its final OD be about 6 hours (OD of 0.3,37 degree cultivation in the M9 liquid nutrient medium containing tsiklomitsin, penbritin, paraxin ₆₀₀=0.6) after adding D type Methionin and isopropylthiogalactoside (IPTG, 100mM) 37 degree to induce 30 hours after, centrifugal collection thalline;

(2) by the sodium phosphate buffer of the PH8.0 of the thalline of centrifugal collection and 0.05M, according to mass volume ratio, be dilution in 1: 5, the high pressure fragmentation, centrifugal collection supernatant, utilize the method for affinity chromatography and anion-exchange chromatography to carry out purifying and can obtain pure containing the amino acid whose Diketoreductase mutant of D type.

In technique scheme, described D type amino acid is D type Methionin.

In technique scheme, described di-carbonyl reduction enzyme refers to the disclosed di-carbonyl reduction enzyme of Chinese invention patent that the patent No. is 200710135391.8.

The contriver cultivates above-mentioned enterobacteria prokaryotic expression system in the M9 substratum that contains D type Methionin, finds 1.2,2.4,3.6,5mM D type Methionin can not cause toxicity can promote to a certain extent thalli growth on the contrary to thalline; In addition, while along with D type amino acid concentration, by 1.2mM, being increased to 5mM, the chlorampenicol resistant of thalline also increases; Wherein, when in substratum, the amount of D type Methionin is 3.6mM, the anti-paraxin of thalline the highest (340 μ g/ml); Anti-paraxin lower (120 μ g/ml) while not adding D type Methionin in substratum.These two experiment preliminary proofs this alpha-non-natural amino acid drawing-in system introducing D type Methionin of fix a point in albumen; The contriver is by isopropylthiogalactoside (IPTG subsequently, 100 μ M) to this thalline induce, the high pressure fragmentation, the centrifuging and taking supernatant, utilize the method for affinity chromatography and anion-exchange chromatography to carry out purifying and can obtain the pure Diketoreductase mutant containing D type Methionin.

Diketoreductase mutant after purifying was 110 ℃ of hydrolysis of 10N HCl 24 hours, the rotation evaporate to dryness, 0.05M the sodium phosphate buffer of PH8.0 dissolves, with upper chirality high-efficient liquid phase analysis after fluorescence derivation reagent N BD-F derivatize, with wild-type di-carbonyl reduction enzyme negative control, compare, the Diketoreductase mutant hydrolyzate can detect the peak of obvious D type Methionin.Through calculated by peak area, the insertion amount of D type Methionin is 115 ± 3%.

The present invention is applicable to other sites simultaneously and introduces, and only any site mutation of the expressing gene of albumen need be become to the TAG codon can realize the introducing at other site D type Methionin.For example, 222 introducing D type Methionins at di-carbonyl reduction enzyme, only need simply by pETDuet-T2 ^dthe TGG codon rite-directed mutagenesis of 222 of the di-carbonyl reduction enzyme gene in K-DKR becomes the TAG codon, utilizes operation described in the present invention can obtain the mutant of the di-carbonyl reduction enzyme that contains D type Methionin in 222 sites.The present invention is applicable to introduce D type Methionin in other albumen simultaneously, only need be by pETDuet-T2 ^din K-DKR, the di-carbonyl reduction enzyme Gene Replacement of first multiple clone site becomes the expressing gene of the albumen of required introducing D type Methionin to get final product.For example, as need be in E.C. 2.3.1.28, introduced D type Methionin, only need be by pETDuet-T2 ^din K-DKR, the gene order of di-carbonyl reduction enzyme replaces to the gene order of E.C. 2.3.1.28, and wanting the codon mutation of introducing D type Methionin position to become the TAG codon, utilize operation described in the present invention can obtain the mutant of the E.C. 2.3.1.28 that contains D type Methionin.

Therefore, a kind of simultaneously claimed method of introducing D type Methionin in target protein of the present invention comprises the following steps:

(1) cut in the certain enzyme of expressivity plasmid the expressing gene that multiple clone site has been introduced target protein, and the purpose site mutation of described expressing gene is become to the TAG codon; Cutting multiple clone site in another certain enzyme of this expressivity plasmid introduces phthe sequence of aminoacyl-tRNA synthetase, obtain recombinant plasmid; 2920 of pACYC184 are replaced to 3542 bit sequences phthe sequence of aminoacyl-tRNA synthetase, 1425 replace to 3 inhibition tRNA sequences to 1524 bit sequences, obtain pAC-ph △-AK3 plasmid; In the host, obtain expression system by pAC-ph △-AK3 plasmid and recombinant plasmid cotransfection;

(2) add D type Methionin in substratum, induce above-mentioned expression system to express target protein, purifying obtains target protein.

In technique scheme, described host is selected from but is not limited to: bacterium, yeast, vegetable cell, zooblast.

Strategy involved in the present invention, method and technology are applicable to the natural or alpha-non-natural amino acid of other D-type simultaneously, and the protein of introducing in source, the aspects such as size, character and structure are all unrestricted.Its strategy comprises: (1) utilizes and add certain density D type Methionin in the substratum of cultivating escherichia coli prokaryotic expression system of the present invention, detect its growing state, (2) monitoring is at the chlorampenicol resistant of escherichia coli prokaryotic expression system of the present invention.

Because technique scheme is used, the present invention compared with prior art has following advantages:

1. method of the present invention is applicable in the protein of arbitrary size introduce D type Methionin; Can introduce D type Methionin in any residue location fixes of protein; D type Methionin is introduced efficiency and is approached 100%; Therefore the method has general applicability, all unrestricted to D type amino acid and the proteins and peptides introduced.

2. the required D type of the method Methionin is cheap, and cost is lower.

The accompanying drawing explanation

Fig. 1 is the impact of 5mM D type Methionin on pAC-ph △-AK3 transformed bacteria growth in the embodiment of the present invention one;

Fig. 2 is in the embodiment of the present invention one 1.2,2.4,3.6mM D type Methionin is on the impact of pAC-ph △-AK3 transformed bacteria growth;

Fig. 3 is in the embodiment of the present invention two 1.2,2.4,3.6,5mM D type Methionin is on the impact of pAC-ph △-AK3 transformed bacteria paraxin tolerance;

Fig. 4 contains the expression and purification SDS-PAGE collection of illustrative plates of the Diketoreductase mutant of D type Methionin in the embodiment of the present invention three;

Fig. 5 contains acid hydrolysis and the analysis of derivative Chiral HPLC of the Diketoreductase mutant of D type Methionin in the embodiment of the present invention five.

Embodiment

Below in conjunction with drawings and Examples, the invention will be further described:

Embodiment mono-: D type Methionin is on the impact containing pAC-ph △-AK3 transformed bacteria growth

At first prepare a kind of escherichia coli prokaryotic expression system, described escherichia coli prokaryotic expression system contains pAC-ph △ with the p15A replicon-AK3 plasmid and with the pETDuet-T2 of ColE1 replicon ^dthe K-DKR plasmid.

In technique scheme, pAC-ph △-AK3 plasmid is obtained by pACYC184 transformation,, with 2920 of p15A replicon: pACYC184, to 3542 bit sequences, replace to phthe sequence of aminoacyl-tRNA synthetase, 1425 replace to 3 inhibition tRNA sequences to 1524 bit sequences.PAC-ph △-AK3 can encode and derive from pyrococcus horikoshiilysyl tRNA synthetic enzyme and inhibition type tRNA(tRNA ^lys) molecule pair, wherein lysyl tRNA synthetic enzyme is by glutamine-tRNA synthetase promotor and terminator regulating and expressing, and inhibition type tRNA is by lpp promotor and rrnC terminator regulating and expressing; In addition, 112 of E.C. 2.3.1.28 aspartic acid codon mutations become amber codon TAG to detect pyrococcus horikoshiilysyl tRNA synthetic enzyme and the right expression of tRNA molecule.Particularly, pAC-ph △-AK3 plasmid has the described nucleotide sequence of SEQ ID No.1.

In technique scheme, pETDuet-T2 ^dk-DKR is obtained by plasmid pETDuet-1 transformation: introduced the sequence of Diketoreductase mutant in first multiple clone site, and 106 to 143 sequences that replace to Diketoreductase mutant, second multiple clone site introduced phthe sequence of aminoacyl-tRNA synthetase, replace and become to 354 bit sequences for 298 phthe sequence of aminoacyl-tRNA synthetase.PETDuet-T2 ^dfirst multiple clone site of K-DKR is connected with the DKR gene of the second bit strip amber mutation; Second multiple clone site is connected with pyrococcus horikoshiilysyl tRNA synthetic enzyme ( phtRNARS) gene; Two fragment genes are all started by T7promoter and are subject to the lactose operon regulating and expressing.Preferably, described pETDuet-T2 ^dthe K-DKR plasmid comprises following four characteristics: second codon of gene of the di-carbonyl reduction enzyme of (1) coding becomes amber codon TAG by the ACC sudden change, (2) N of di-carbonyl reduction enzyme mutant gene end has added the label of His6, (3) mutator gene of di-carbonyl reduction enzyme is building up to first multiple clone site of pETDuet-1, and under T7promoter starts by the lactose operon regulating and expressing.(4) second of pETDuet-1 multiple clone site added phthe gene of tRNARS strengthens phthe expression of tRNARS is to improve the right suppression efficiency of lysyl tRNA synthetic enzyme molecule.Particularly, pETDuet-T2 ^dthe K-DKR plasmid has the described nucleotide sequence of SEQ ID No.2.

PACYC184 and pETDuet-1 are commercial carriers, the U.S. Patent Application Publication specification sheets that the sequence of tRNA and tRNA synthetic enzyme can be US 2006/0177900 referring to publication number, and particularly, the sequence of tRNA refers to shown in SEQ ID No.3; The sequence of tRNA synthetic enzyme refers to shown in SEQ ID No.4.

In technique scheme, described intestinal bacteria are e. coli bl21 (DE3).

In technique scheme, described escherichia coli prokaryotic expression system is by pAC-ph △-AK3 plasmid and pETDuet-T2 ^dk-DKR plasmid co-transfection e. coli bl21 (DE3) cell obtains.

By pAC-ph △-AK3 and pETDuet-T2 ^dtwo plasmid co-transfection e. coli bl21s of K-DKR (DE3) competent cell, coat on the flat board of the paraxin that contains 4 μ g/ml tsiklomitsins, 20 μ g/ml, cultivate approximately 30 hours for 37 ℃, the single bacterium colony on picking cotransformation flat board in the LB liquid nutrient medium with tsiklomitsin and paraxin, cultivate 20 hours, get the LB nutrient solution and be diluted in the M9 substratum with tsiklomitsin and paraxin and make its OD ₆₀₀be about 0.1,37 ℃ and cultivate approximately 30 hours, be transferred to containing 1.2,2.4,3.6, the D type Methionin of 5mM, contain 2 kinds of antibiotic M9 substratum approximately 40 hours, during got nutrient solution every 2 hours and survey OD ₆₀₀value.From growth curve (Fig. 1, Fig. 2) can find out 3.6, the D type Methionin of 5mM can promote at logarithmic phase the growth of cell, and the D type Methionin of 1.2mM and 2.4mM can not suppress thalli growth.Spherical broken line in Fig. 1 represents that the D type Methionin of 5mM transforms intestinal bacteria to pAC-ph △-AK3, square broken line represents blank, square broken line in Fig. 2 represents that the D type Methionin of 3.6 mM transforms intestinal bacteria to pAC-ph △-AK3, spherical broken line graph represents that the D type Methionin of 1.2M transforms intestinal bacteria to pAC-ph △-AK3, and the rhombus broken line graph represents that the D type Methionin of 2.4M transforms intestinal bacteria to pAC-ph △-AK3.

Embodiment bis-: D type Methionin is on the impact containing pAC-ph △-AK3 transformed bacteria paraxin tolerance.

By pAC-ph △-AK plasmid transfection e. coli bl21 (DE3) competent cell, coat containing 4 μ g/ml tsiklomitsins, 20 μ g/ml flat board on, cultivate approximately 30 hours for 37 ℃, the single bacterium colony on picking cotransformation flat board in the LB liquid nutrient medium with tsiklomitsin, paraxin, cultivate 20 hours, get the LB nutrient solution and be diluted in the M9 substratum with tsiklomitsin, paraxin and make its OD ₆₀₀be about 0.2, cultivate approximately 30 hours for 37 ℃, under the D type Methionin that contains different concns gradient (1.2,2.4,3.6,5mM), being transferred in the M9 substratum of chloramphenicol concentration gradient of 0,20,40,60,80,100,120,140,160,180,200,220,240,260,280,300,320,340,360 μ g/ml, is not wherein control group containing paraxin.Cultivate and approximately after 30 hours, measure its OD ₆₀₀value, calculate.Suppress curve from paraxin and can find out (as shown in Figure 3), the paraxin tolerance of thalline increases and strengthens along with the D type lysine concentration in substratum.Thus, we may safely draw the conclusion, and lysyl tRNA synthetic enzyme and inhibition tRNA have very high selectivity to D type Methionin in the M9 substratum.

Embodiment tri-: containing the expression and purification of D type Methionin Diketoreductase mutant

The fixed point drawing-in system of D type Methionin is by including pAC-ph △-AK3 and pETDuet-T2 ^dthe cotransformation e. coli bl21 (DE3) of two plasmids of K-DKR forms.Two plasmids are respectively with compatible replicon p15A and ColE1, and wherein pAC-ph △-AK3 coding derives from pyrococcus horikoshiilysyl tRNA synthetic enzyme ( phtRNARS) and inhibition tRNA, pETDuet-T2 ^dthe mutator gene that K-DKR contains di-carbonyl reduction enzyme, wherein pETDuet-T2 ^dthe K-DKR plasmid comprises following four characteristics: second codon of gene of the di-carbonyl reduction enzyme of (1) coding becomes amber codon TAG by the ACC sudden change, (2) N of di-carbonyl reduction enzyme mutant gene end has added the label of His6, (3) mutator gene of di-carbonyl reduction enzyme is building up to first multiple clone site of pETDuet-1, and under T7promoter starts by the lactose operon regulating and expressing.(4) second of pETDuet-1 multiple clone site added phthe gene of tRNARS strengthens phthe expression of tRNARS is to improve the right suppression efficiency of lysyl tRNA synthetic enzyme molecule.By pAC-ph △-AK3 and pETDuet-T2 ^dk-DKR plasmid co-transfection e. coli bl21 (DE3) cell, flat board is coated containing 4 μ g/ml tsiklomitsins, the penbritin of 40 μ g/ml, on the LB flat board of the paraxin of 20 μ g/ml, 37 degree are cultivated approximately 20 hours, picking list bacterium colony is in containing tsiklomitsin, penbritin, LB liquid nutrient medium 37 degree of paraxin are cultivated and are approximately transferred after 20 hours in containing tsiklomitsin, penbritin, the M9 liquid nutrient medium of paraxin makes its final OD be about 0.1, 37 degree are cultivated 30 hours, transfer in containing tsiklomitsin, penbritin, the M9 liquid nutrient medium of paraxin makes its final OD be about 0.3, 37 degree are cultivated 6 hours (OD ₆₀₀=0.6) after adding D type Methionin and IPTG (100mM) 37 degree to induce 30 hours after, centrifugal collection thalline.

By the sodium phosphate buffer of the PH8.0 of the thalline of centrifugal collection and 0.05M, according to mass volume ratio, be the 1:5 dilution, high pressure fragmentation, centrifugal collection supernatant.After the upper cleer and peaceful Ni-NTA post material 4 degree Static Adsorption of 25ml are spent the night, first use 10 column volumes of broken buffer solution elution, adopt subsequently containing 10,50,100,200, the 0.05M PH8.0 sodium phosphate buffer of 500mM imidazoles 5 column volumes of wash-out respectively, collection 100,200mM imidazoles elution fraction, concentrated, sampling is surveyed enzyme and is lived, and SDS-PAGE detects.Nickel post wash-out is collected to anion-exchange chromatography purifying on liquid, and its eluent components is: the sodium phosphate buffer of the PH8.0 of A pump: 0.05M, the sodium phosphate buffer of the pH8.0 of B pump: 0.05M is containing the NaCl of 1M.Uv-absorbing by UV-detector monitoring elutriant at 280nm.Elution requirement is: 15 column volumes of 0-30% salt concn linear gradient elution, when salt concn is 14.2%, target protein is by wash-out.Collect the target protein elutriant, concentrated, sampling is surveyed enzyme and is lived, and SDS-PAGE detects.Purification result is as shown in Figure 4: swimming lane 1 is standard protein Marker, and swimming lane 2 is for inducing rear bacterial cell disruption supernatant, the elutriant of swimming lane 3 after for the Ni-NTA purifying after concentrated, and swimming lane 4 is received elutriant for the anion-exchange chromatography purifying after concentrated.

Embodiment tetra-: the acid hydrolysis and the derivatize that contain the Diketoreductase mutant of D type Methionin

Get 3mg containing the 110 degree hydrolysis 24 hours in 30ml 10N hydrochloric acid of the Diketoreductase mutant albumen of D type Methionin, the rotation evaporate to dryness, be dissolved in the sodium phosphate buffer (also can be the sodium borate buffer liquid of the PH 8.0 of 0.1M) of 500 μ l PH 8.0.Get 50 μ l lysates and 30 μ l 50mM NBD-F solution and mix, 60 degree heating add 1% acetic acid ethanolic soln 920 μ l termination reactions after 5 minutes.

Embodiment five: containing the Chiral HPLC analysis of the NBD-F derivative of the Diketoreductase mutant hydrolysate of D type Methionin

The high performance liquid phase instrument is Shimadzu SCL-2010A, and the chiral analysis post is the OD-RH chiral column, and mobile phase composition is: the A pump is the distilled water containing 1 ‰ TFA, and the B pump is the acetonitrile containing 1 ‰ TFA.The detection wavelength of UV-detector is 470nm.The B pump linear gradient elution of elution requirement: 35%-40% after 150 minutes the 40%B pump maintain 10min.As shown in Figure 5, and D, the L-type lysine derivative is compared, and within 113.3 minutes, occurs the peak of D type Methionin in Diketoreductase mutant proteolysis derivative collection of illustrative plates.And the peak of D type Methionin does not appear at the control group of wild-type mutant protein.Proof D type Methionin only inserts in the second position fixed point of di-carbonyl reduction enzyme.Through the peak area of calculated by peak area contrast L-type Methionin and D type Methionin, D type Methionin is 115 ± 3% in deputy insertion amount.

Nucleotide and/or aminoacid sequence table

<110 > China Medicine University

<120 > a kind of preparation method of the mutant that contains the amino acid whose di-carbonyl reduction enzyme of D type

<160> 4

<170> PatentIn version 3.5

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<211> 5447

<212> DNA

<213 > synthetic

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aaaacgatct caagaagatc atcttattaa tcagataaaa tatttggtaa gatctcgaac 1440

gatcaaaaat aagtgccttc ccatcaaaaa aatattctca acataaaaaa ctttgtgtaa 1500

tacttgtaac gctacatgga gattaactca atctagctag aggaattcgg gcccgagcca 1560

gccggagagc ggcgggccaa acccgcaggc gcgggcaaac ccgccgggcc cgccactgca 1620

gcttcgtgtc atccttagcg aaagctaagg attttttttg catgcaaccg gtaagatctc 1680

gaacgatcaa aaataagtgc cttcccatca aaaaaatatt ctcaacataa aaaactttgt 1740

gtaatacttg taacgctaca tggagattaa ctcaatctag ctagaggaat tcgggcccga 1800

gccagccgga gagcggcggg ccaaacccgc aggcgcgggc aaacccgccg ggcccgccac 1860

tgcagcttcg tgtcatcctt agcgaaagct aaggattttt tttgcatgca accggtaaga 1920

tctcgaacga tcaaaaataa gtgccttccc atcaaaaaaa tattctcaac ataaaaaact 1980

ttgtgtaata cttgtaacgc tacatggaga ttaactcaat ctagctagag gaattcgggc 2040

ccgagccagc cggagagcgg cgggccaaac ccgcaggcgc gggcaaaccc gccgggcccg 2100

ccactgcagc ttcgtgtcat ccttagcgaa agctaaggat tttttttgca tgcaaccagc 2160

tttaatgcgg tagtttatca cagttaaatt gctaacgcag tcaggcaccg tgtatgaaat 2220

ctaacaatgc gctcatcgtc atcctcggca ccgtcaccct ggatgctgta ggcataggct 2280

tggttatgcc ggtactgccg ggcctcttgc gggatatcgt ccattccgac agcatcgcca 2340

gtcactatgg cgtgctgcta gcgctatatg cgttgatgca atttctatgc gcacccgttc 2400

tcggagcact gtccgaccgc tttggccgcc gcccagtcct gctcgcttcg ctacttggag 2460

ccactatcga ctacgcgatc atggcgacca cacccgtcct gtggatcctc tacgccggac 2520

gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca 2580

tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg 2640

gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat 2700

tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg 2760

agtcgcataa gggagagcgt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct 2820

tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc 2880

aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct 2940

ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc 3000

aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg 3060

gcatggcggc cgacgcgctg ggctacgtct tgctggcgtt cgcgacgcga ggctggatgg 3120

ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca 3180

tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg ctcgcggctc 3240

ttaccagcct aacttcgatc actggaccgc tgatcgtcac ggcgatttat gccgcctcgg 3300

cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt gtctgcctcc 3360

ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca 3420

cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt 3480

gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg 3540

cacgcggcgc atgggcccta taagatcata cgccgttata cgttgtttac gctttgagga 3600

atcggtacca tggttcattg ggccgattat attgctgata aaataattag agagaggggg 3660

gagaaggaga agtacgttgt tgagagtgga ataacgccaa gtggttacgt tcacgttggg 3720

aactttaggg agctttttac agcttatatt gtgggccatg ccctaaggga taaggggtat 3780

gaggttaggc acatccacat gtgggatgat tatgatagat ttaggaaggt tccaaggaac 3840

gttccccagg aatggaaaga ttacctggga atgcccatta gtgaagttcc tgatccctgg 3900

ggatgccatg agagttatgc tgaacacttc atgagaaagt tcgaggagga ggtagaaaaa 3960

ttagggatcg aagttgactt tctttatgcg agtgaactct acaagagagg ggaatattct 4020

gaggagataa ggttagcctt tgagaaaagg gataagataa tggagatact aaacaagtat 4080

agggaaattg cgaaacaacc tccccttcca gagaactggt ggcccgcaat ggtttactgc 4140

cctgagcata ggagggaagc agagatcatt gaatgggatg ggggctggaa ggttaagtat 4200

aagtgccccg aaggtcacga gggatgggtt gatataagga gtgggaacgt gaaactgagg 4260

tggcgtgttg attggcccat gcgttggtct cactttggcg ttgacttcga acctgctgga 4320

aaggatcatc ttgtggctgg ttcaagctac gatacgggaa aggagattat aaaggaagtt 4380

tatggaaagg aagctccgtt atctttaatg tatgagtttg ttggaattaa ggggcagaag 4440

gggaagatga gtggtagtaa gggaaatgtt attttactca gcgatctgta tgaggttctt 4500

gagccaggtc tcgttagatt tatctacgct cggcataggc caaacaagga gataaagata 4560

gatctaggtc ttggcattct aaacctctac gatgagttcg ataaagttga gagaatatac 4620

ttcggggttg agggtggtaa aggtgatgat gaagaattaa ggaggactta cgagctttca 4680

taaagaaaca gcaaacaatc caaaacgccg cgttcagcgg cgttttttct gcttttctac 4740

gcgtgatcat atcgtcaatt attacctcca cggggagagc ctgagcaaac tggcctcagg 4800

catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat 4860

agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg aatttgcttt 4920

cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcacc aggcgtttaa 4980

gggcaccaat aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt 5040

tgtaattcat taagcattct gccgacatgg aagccatcac agacggcatg atgaacctga 5100

atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg 5160

ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag 5220

ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt 5280

tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg 5340

tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg 5400

tgaacactat cccatatcac cagctcaccg tctttcattg ccatacg 5447

<210> 2

<211> 7252

<212> DNA

<213 > synthetic

<400> 2

ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag 60

gagatatacc atgggcagca gccatcacca tcatcaccac agccagatga ccggcatcac 120

gaatgtcacc gttctcggaa ccggcgtgct gggttcgcag atcgcgttcc agaccgcgtt 180

ccacggcttc gccgtcaccg cgtacgacat caacaccgac gcgctcgacg ccgccaagaa 240

gcgcttcgag ggcctcgcgg ccgtgtacga gaaggaggtc gcgggcgctg cagacggcgc 300

cgcgcagaag gcgctgggcg gcatccgcta ctccgacgac ctggcgcagg ccgtgaagga 360

cgccgacctc gtcatcgagg ccgtgccgga gagcctcgac ctcaagcgcg acatctacac 420

gaagctgggc gagctggcgc cggcgaagac gatcttcgcc accaactcct ccaccctgct 480

gccgagcgac ctggtcggct acaccggccg gggcgacaag ttcctcgccc tgcacttcgc 540

caaccacgtg tgggtgaaca acaccgccga ggtgatgggc acgacgaaga ccgaccccga 600

ggtgtaccag caggtcgtcg agttcgcctc cgcgatcggg atggtgccga tcgagctgaa 660

gaaggagaag gcgggctacg tgctcaactc cctcctcgtc ccgctgctgg atgcggcggc 720

cgagctgctg gtggacggca tcgccgaccc cgagacgatc gacaagacgt ggcgcatcgg 780

cacgggcgca cccaagggtc cgttcgagat cttcgacatc gtcggcctca cgacggccta 840

caacatctcc tcggtctccg gccccaagca gcgcgagttc gccgcgtacc tcaaggaaaa 900

ctacatcgac aaaggcaagc tgggcctcgc gaccggcgag ggcttctacc ggtactgaag 960

cttgcggccg cataatgctt aagtcgaaca gaaagtaatc gtattgtaca cggccgcata 1020

atcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt ccccatctta 1080

gtatattagt taagtataag aaggagatat acatggttca ttgggccgat tatattgctg 1140

ataaaataat tagagagagg ggggagaagg agaagtacgt tgttgagagt ggaataacgc 1200

caagtggtta cgttcacgtt gggaacttta gggagctttt tacagcttat attgtgggcc 1260

atgccctaag ggataagggg tatgaggtta ggcacatcca catgtgggat gattatgata 1320

gatttaggaa ggttccaagg aacgttcccc aggaatggaa agattacctg ggaatgccca 1380

ttagtgaagt tcctgatccc tggggatgcc atgagagtta tgctgaacac ttcatgagaa 1440

agttcgagga ggaggtagaa aaattaggga tcgaagttga ctttctttat gcgagtgaac 1500

tctacaagag aggggaatat tctgaggaga taaggttagc ctttgagaaa agggataaga 1560

taatggagat actaaacaag tatagggaaa ttgcgaaaca acctcccctt ccagagaact 1620

ggtggcccgc aatggtttac tgccctgagc ataggaggga agcagagatc attgaatggg 1680

atgggggctg gaaggttaag tataagtgcc ccgaaggtca cgagggatgg gttgatataa 1740

ggagtgggaa cgtgaaactg aggtggcgtg ttgattggcc catgcgttgg tctcactttg 1800

gcgttgactt cgaacctgct ggaaaggatc atcttgtggc tggttcaagc tacgatacgg 1860

gaaaggagat tataaaggaa gtttatggaa aggaagctcc gttatcttta atgtatgagt 1920

ttgttggaat taaggggcag aaggggaaga tgagtggtag taagggaaat gttattttac 1980

tcagcgatct gtatgaggtt cttgagccag gtctcgttag atttatctac gctcggcata 2040

ggccaaacaa ggagataaag atagatctag gtcttggcat tctaaacctc tacgatgagt 2100

tcgataaagt tgagagaata tacttcgggg ttgagggtgg taaaggtgat gatgaagaat 2160

taaggaggac ttacgagctt tcataatcga gtctggtaaa gaaaccgctg ctgcgaaatt 2220

tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg ctgctgccac 2280

cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 2340

gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta 2400

agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 2460

cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 2520

gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 2580

aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 2640

cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 2700

acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 2760

tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 2820

acgtttacaa tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga 2880

tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 2940

ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 3000

catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 3060

ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 3120

caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 3180

ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 3240

tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 3300

cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 3360

aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 3420

tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 3480

gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 3540

cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 3600

aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 3660

tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 3720

tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 3780

gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt 3840

tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 3900

ataggtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 3960

agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 4020

aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 4080

ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 4140

gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 4200

aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 4260

aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 4320

gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 4380

aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 4440

aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 4500

cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 4560

cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 4620

tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 4680

tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 4740

ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 4800

ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat 4860

acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg 4920

ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 4980

tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc 5040

tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg 5100

cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca 5160

tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt 5220

tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg 5280

atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc 5340

gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg 5400

ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg 5460

ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg 5520

ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt 5580

cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca 5640

cgatcatgct agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 5700

agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc 5760

gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 5820

aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt 5880

gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg 5940

tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata 6000

taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc 6060

agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc 6120

atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg 6180

gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta 6240

tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg 6300

atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg 6360

gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca 6420

ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc 6480

agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg 6540

cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc 6600

gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc 6660

aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc 6720

atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg 6780

cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt 6840

ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag 6900

gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat 6960

taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc 7020

atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc 7080

gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc 7140

gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc 7200

gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta ta 7252

<210> 3

<211> 254

<212> DNA

<213 > synthetic

<400> 3

ggtaagatct cgaacgatca aaaataagtg ccttcccatc aaaaaaatat tctcaacata 60

aaaaactttg tgtaatactt gtaacgctac atggagatta actcaatcta gctagaggaa 120

ttcgggcccg uagcucagcc ugguagagcg gcgggcucua aacccgcagg ucgcggguuc 180

aaaucccgcc gggcccgcca ctgcagcttc gtgtcatcct tagcgaaagc taaggatttt 240

ttttgcatgc aacc 254

<210> 4

<211> 1192

<212> DNA

<213 > synthetic

<400> 4

gggccctata agatcatacg ccgttatacg ttgtttacgc tttgaggaat cggtaccatg 60

gttcattggg ccgattatat tgctgataaa ataattagag agagggggga gaaggagaag 120

tacgttgttg agagtggaat aacgccaagt ggttacgttc acgttgggaa ctttagggag 180

ctttttacag cttatattgt gggccatgcc ctaagggata aggggtatga ggttaggcac 240

atccacatgt gggatgatta tgatagattt aggaaggttc caaggaacgt tccccaggaa 300

tggaaagatt acctgggaat gcccattagt gaagttcctg atccctgggg atgccatgag 360

agttatgctg aacacttcat gagaaagttc gaggaggagg tagaaaaatt agggatcgaa 420

gttgactttc tttatgcgag tgaactctac aagagagggg aatattctga ggagataagg 480

ttagcctttg agaaaaggga taagataatg gagatactaa acaagtatag ggaaattgcg 540

aaacaacctc cccttccaga gaactggtgg cccgcaatgg tttactgccc tgagcatagg 600

agggaagcag agatcattga atgggatggg ggctggaagg ttaagtataa gtgccccgaa 660

ggtcacgagg gatgggttga tataaggagt gggaacgtga aactgaggtg gcgtgttgat 720

tggcccatgc gttggtctca ctttggcgtt gacttcgaac ctgctggaaa ggatcatctt 780

gtggctggtt caagctacga tacgggaaag gagattataa aggaagttta tggaaaggaa 840

gctccgttat ctttaatgta tgagtttgtt ggaattaagg ggcagaaggg gaagatgagt 900

ggtagtaagg gaaatgttat tttactcagc gatctgtatg aggttcttga gccaggtctc 960

gttagattta tctacgctcg gcataggcca aacaaggaga taaagataga tctaggtctt 1020

ggcattctaa acctctacga tgagttcgat aaagttgaga gaatatactt cggggttgag 1080

ggtggtaaag gtgatgatga agaattaagg aggacttacg agctttcata aagaaacagc 1140

aaacaatcca aaacgccgcg ttcagcggcg ttttttctgc ttttctacgc gt 1192

Claims (7)

1. an escherichia coli prokaryotic expression system, is characterized in that, described escherichia coli prokaryotic expression system contains pAC-ph △ with the p15A replicon-AK3 plasmid and with the pETDuet-T2 of ColE1 replicon ^dthe K-DKR plasmid; PAC-ph △-AK3 plasmid is as shown in nucleotide sequence as described in SEQ ID No.1; PETDuet-T2 ^dthe K-DKR plasmid is as shown in nucleotide sequence as described in SEQ ID No.2.

2. escherichia coli prokaryotic expression system according to claim 1, is characterized in that, pAC-ph △-AK3 plasmid is obtained by the pACYC184 transformation: 2920 of pACYC184 replace to 3542 bit sequences phthe sequence of aminoacyl-tRNA synthetase, 1425 replace to 3 inhibition tRNA sequences to 1524 bit sequences, lysyl tRNA synthetic enzyme is by glutamine-tRNA synthetase promotor and terminator regulating and expressing, and inhibition type tRNA is by lpp promotor and rrnC terminator regulating and expressing.

3. escherichia coli prokaryotic expression system according to claim 1, is characterized in that pETDuet-T2 ^dk-DKR is obtained by plasmid pETDuet-1 transformation: the sequence of having introduced Diketoreductase mutant in first multiple clone site, 106 to 143 sequences that replace to Diketoreductase mutant, second codon of gene of the di-carbonyl reduction enzyme of coding becomes amber codon TAG by the ACC sudden change, and second multiple clone site introduced phthe sequence of aminoacyl-tRNA synthetase, replace and become to 354 bit sequences for 298 phthe sequence of aminoacyl-tRNA synthetase.

4. escherichia coli prokaryotic expression system according to claim 1, is characterized in that, described intestinal bacteria are preferably e. coli bl21 (DE3).

5. escherichia coli prokaryotic expression system according to claim 1, is characterized in that, described escherichia coli prokaryotic expression system is by pAC-ph △-AK3 plasmid and pETDuet-T2 ^dk-DKR plasmid co-transfection e. coli bl21 (DE3) cell obtains.

6. the method for the Diketoreductase mutant that utilizes the described enterobacteria prokaryotic expression system preparation of claim 1 to contain D type Methionin, is characterized in that, comprises the following steps:

(1) add D type Methionin in substratum, isopropylthiogalactoside induces the described escherichia coli prokaryotic expression system of claim 1 to express, high pressure fragmentation, centrifuging and taking supernatant;

(2) adopt the method purifying supernatant liquor of affinity chromatography and anion-exchange chromatography combination to obtain containing the amino acid whose di-carbonyl reduction enzyme of D type.

7. a method of introducing D type Methionin in di-carbonyl reduction enzyme, is characterized in that, comprises the following steps:

(1) cut in the certain enzyme of expressivity plasmid the expressing gene that multiple clone site has been introduced target protein, and the purpose site mutation of described expressing gene is become to the TAG codon; Cutting multiple clone site in another certain enzyme of this expressivity plasmid introduces phthe sequence of aminoacyl-tRNA synthetase, obtain recombinant plasmid pETDuet-T2 ^dk-DKR; 2920 of pACYC184 are replaced to 3542 bit sequences phthe sequence of aminoacyl-tRNA synthetase, 1425 replace to 3 inhibition tRNA sequences to 1524 bit sequences, obtain pAC-ph △-AK3 plasmid; In the host, obtain expression system by pAC-ph △-AK3 plasmid and recombinant plasmid cotransfection; PAC-ph △-AK3 plasmid is as shown in nucleotide sequence as described in SEQ ID No.1; PETDuet-T2 ^dthe K-DKR plasmid is as shown in nucleotide sequence as described in SEQ ID No.2;

(2) add D type Methionin in substratum, induce above-mentioned expression system to express target protein, purifying obtains target protein.

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