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Polypeptide, DNA molecule, recombination carrier, transformant, application of polypeptide, application of DNA molecule and application of transformant

A DNA molecule and recombinant vector technology, applied in the biological field, can solve problems such as serious environmental pollution, and achieve the effect of high optical purity and high yield

Active Publication Date: 2019-04-02
安徽瑞达健康产业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In summary, the prior art has the following problems: in the process of preparing the intermediate of pregabalin (the intermediate is (R)-3-isobutylglutaric acid monoamide), the existing chemical resolution method needs to use To a large amount of chloroform, serious environmental pollution

Method used

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  • Polypeptide, DNA molecule, recombination carrier, transformant, application of polypeptide, application of DNA molecule and application of transformant
  • Polypeptide, DNA molecule, recombination carrier, transformant, application of polypeptide, application of DNA molecule and application of transformant
  • Polypeptide, DNA molecule, recombination carrier, transformant, application of polypeptide, application of DNA molecule and application of transformant

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Experimental program
Comparison scheme
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specific Embodiment approach

[0095] 1. Polypeptide

[0096] Amino acid sequence deletion, insertion, substitution or addition of one or several amino acids can be introduced by site-directed mutation (Nucleic Acids Res (1982) 10: 6487; Methods in Enzymol (1983) 100: 448; Molecular Cloning2nd Edt., Cold Spring Harbor Laboratory Press (1989); PCR A Practical Approach, IRLP press (1991) pp. 200) and other known methods or other methods to introduce into the amino acid sequence. The number of "several amino acids" may be, for example, 30 or less, preferably 20 or less, more preferably 10 or less, further preferably 7 or less, particularly preferably 5 or less.

[0097] When two amino acid sequences were compared and analyzed using the homology search program BLAST (W.R.Pearson & D.J. Lipman P.N.A.S. (1988) 85: 2444-2448), the sequence identity was expressed as an identity (Identity) value with respect to the entire sequence.

[0098] The polypeptide of the present invention can be obtained by chemical synthe...

Embodiment 1

[0131] Strain Construction of Hydantoinase Recombinant Transformant

[0132] The hydantoinase sequences derived from Jannaschia CCS1 strain, Jannaschia sp.EhC01 strain, Jannaschia aquimarina strain, Litoreibacter ponti strain, Ralstonia pickettii strain, and Arthrobacter aurescens strain were searched on NCBI, and the codons were optimized in the E. coli expression system in turn. (Using the conventional online website http: / / www.jcat.de / for optimization), the optimized gene was fully synthesized. A BamHI restriction site was added to the N-terminus of each gene, an XhoI restriction site was added to the C-terminus, and after restriction digestion, it was connected to the BamHI and XhoI restriction sites of the plasmid pET-28a, with a His tag on the N-terminus. The process is as follows:

[0133] The enzyme digestion system is as follows:

[0134] Target gene / plasmid 1ug

[0135] BamHI 1ul

[0136] XhoI 1ul

[0137] 10×buffer 3ul

[0138] wxya 2 O make up to 30ul

[0...

Embodiment 2

[0168] Catalytic bacteria and protein preparation

[0169] The E.coli RD01 transformant was inoculated in 50ml / 250ml of LB medium (yeast extract 0.5%, peptone 1%, sodium chloride 1%, pH 7.0) containing 50 μg / ml kanamycin, at 37°C, Shake culture at 220rpm, add 0.5mM IPTG to induce when growing to OD600 of 0.6-0.8, the induction condition is 20 degrees, 200rpm. The cells were collected by centrifugation and suspended in 50 mM phosphate buffer (pH 7.0).

[0170] The cells were ultrasonically disrupted and then centrifuged, and the supernatant was passed through pre-filled and equilibrated Ni-NTA (purchased from Solarbio | product number: P2010), and the protein in the supernatant was collected. Impurities were eluted with 20 mM imidazole, and proteins were eluted with 250 mM imidazole. Then use a 30KDa protein concentration tube to concentrate the protein to 2 mg / mL to obtain a protein concentrate.

[0171] Similarly, E.coli RD02, E.coli RD03, E.coli RD04, E.coli RD05 and E.co...

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Abstract

The invention discloses polypeptide, a DNA molecule, a recombination carrier, a transformant, an application of the polypeptide, an application of DNA molecule and an application of the transformant,and belongs to the field of a biological technology. In accordance with the problems that in the prior art, in the process of preparing the intermediate ( the intermediate is(R) 3-carbamoymethyl-5-methylhexanoic acid ) of Pregabalin, a large amount of chloroform is needed in a conventional chemical splitting method, so that the environmental pollution is serious, the invention provides the polypeptide. The polypeptide is used, so that 3-Isobutyl glutarimide can be hydrolyzed to obtain the (R) 3-carbamoymethyl-5-methylhexanoic acid which is high in 3-Isobutyl glutarimide and high in yield.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to polypeptides, DNA molecules, recombinant vectors, transformants and applications thereof. Background technique [0002] Pregabalin is a 3-position substituted analogue of GABA developed by Pfizer. It is a related compound and its intended successor to the antiepileptic drug Gabapentin, which has achieved great commercial success. It was approved by the European Union in July 2004 An add-on therapy drug for the management of peripheral neuropathic pain and for the treatment of some epileptic seizures. Pregabalin has also been approved in the United States at the end of December 2004 for the relief of neuropathic pain associated with diabetic peripheral neuropathy and postherpetic neuralgia, making it the first drug in the United States to be officially approved to simultaneously treat these two diseases. The drug for the treatment of neuropathic pain was approved by the FDA in 2005 f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/86C12N15/55C12N15/70C12N1/21C12P41/00C12P13/02C12P13/00C12R1/19
CPCC12N9/86C12P13/005C12P13/02C12P41/006C12Y305/02002
Inventor 陈雷史实余继刚宣超汤伟强蔡祥鹏汪晓东王淯王娜娜王保如
Owner 安徽瑞达健康产业有限公司
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