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

A D-type, aminoacyl technology, applied in the field of preparing proteins and/or polypeptides containing D-type amino acids, can solve problems such as unsatisfactory, and achieve the effect of low cost and low price

Inactive Publication Date: 2012-06-27
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the genetic code expansion technology can introduce more than 100 kinds of L-type unnatural amino acids into proteins of any size at fixed points, which is a major breakthrough compared with previous technologies, but this technology still cannot satisfy people's desire to modify amino acids at fixed points by changing the chirality of amino acids. and requirements for engineered proteins and peptides

Method used

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  • Preparation method of mutant of double-carbonyl reductase containing D-amino acid
  • Preparation method of mutant of double-carbonyl reductase containing D-amino acid
  • Preparation method of mutant of double-carbonyl reductase containing D-amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Effect of D-lysine on the growth of transformed bacteria containing pAC-ph△-AK3

[0041] Firstly, an E. coli prokaryotic expression system was prepared, which contained pAC-ph△-AK3 plasmid with p15A replicon and pETDuet-T2 with ColE1 replicon D K-DKR plasmid.

[0042] In the above technical scheme, the pAC-ph△-AK3 plasmid is transformed from pACYC184, with a p15A replicon: the sequence from position 2920 to position 3542 of pACYC184 is replaced by pH In the sequence of aminoacyl tRNA synthetase, the sequence from position 1425 to position 1524 is replaced by three inhibitory tRNA sequences. pAC-ph△-AK3 can code from Pyrococcus horikoshii Lysyl tRNA synthetase and inhibitory tRNA (tRNA Lys ) molecular pair, in which lysyl tRNA synthetase is regulated by glutamine tRNA synthetase promoter and terminator, and suppressor tRNA is regulated by lpp promoter and rrnC terminator; in addition, chloramphenicol acetyl transfer The 112th aspartic acid codon of the e...

Embodiment 2

[0048] Example 2: Effect of D-lysine on the chloramphenicol tolerance of pAC-phΔ-AK3-containing transformed bacteria.

[0049] Transfect the pAC-ph△-AK plasmid into Escherichia coli BL21 (DE3) competent cells, spread it on a plate containing 4 μg / ml tetracycline and 20 μg / ml tetracycline, incubate at 37°C for about 30 hours, pick the co-transformation plate A single colony was cultured in LB liquid medium with tetracycline and chloramphenicol for 20 hours, and the LB culture solution was diluted into M9 medium with tetracycline and chloramphenicol to make its OD 600 About 0.2, cultured at 37°C for about 30 hours, under D-lysine with different concentration gradients (1.2, 2.4, 3.6, 5mM), transferred to 0, 20, 40, 60, 80, 100, 120, 140 , 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360 μg / ml of chloramphenicol concentration gradient in the M9 medium, which does not contain chloramphenicol as the control group. Measure the OD after culturing for about 30 hours 600 value, ...

Embodiment 3

[0050] Example 3: Expression and purification of D-type lysine double carbonyl reductase mutant

[0051]The site-directed introduction system of D-lysine is composed of pAC-ph△-AK3 and pETDuet-T2 D K-DKR two plasmids co-transformed Escherichia coli BL21 (DE3) constituted. The two plasmids contain compatible replicons p15A and ColE1 respectively, and the pAC-ph△-AK3 code is derived from Pyrococcus horikoshii Lysyl tRNA synthetase ( pH tRNARS) and inhibitory tRNA, pETDuet-T2 D K-DKR contains a mutant gene of double carbonyl reductase, in which pETDuet-T2 D The K-DKR plasmid contains the following four features: (1) The second codon of the gene encoding the double carbonyl reductase is mutated from ACC to the amber codon TAG, (2) His6 is added to the N-terminal of the double carbonyl reductase mutant gene (3) The mutant gene of double carbonyl reductase was constructed into the first multiple cloning site of pETDuet-1, and its expression was regulated by the lactose operon...

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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 frompyrococcushorikoshii 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

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing proteins and / or polypeptides containing D-type amino acids. Background technique [0002] Introducing unnatural amino acids into proteins and peptides to obtain new properties is a strategy that has been widely used in the fields of protein and peptide drug modification and preparation of biomaterials. Currently, there are three main methods for introducing unnatural amino acids into protein peptides: [0003] (1) Polypeptide solid-phase synthesis method. The peptide solid-phase step-by-step synthesis method first adopted by Merrifield introduced D-amino acid and arginine analogues into luteinizing hormone releasing hormone (see: Science. 1965; 150:178-85.). This method enables the introduction of unnatural amino acids at any position in polypeptides of less than 100 residues. However, it has the disadvantages of being limited to the synthesis of short peptide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/02C12N15/63C12R1/19
Inventor 刘智志杨欣陈依军
Owner CHINA PHARM UNIV
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