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Recombinant expression plasmid based on T7 promoter, and transformant and application thereof

A technology for expressing plasmids and promoters, which can be used in the application of the transformant, in the production of 1,5-pentanediamine, and in the field of fermentation to produce polypeptides such as lysine decarboxylase, which can solve the problem of restricting the expression of target genes In order to achieve the effect of increasing the copy number, preventing protein folding restriction, and convenient use

Pending Publication Date: 2018-08-28
CATHAY R&D CENT CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the above-mentioned induced expression system, there is a problem that the expression intensity is not strong enough, among which tet and ara are weak promoters, which can only express a small amount of protein, enzyme or polypeptide, while lac, lacUV 5 , tac, and trc promoters are medium-strength promoters. If they need to express a large amount of enzymes, proteins or polypeptides, they need to add more inducers, which will increase the cost of inducing expression.
However, this invention greatly limits the amount of expression of the target gene

Method used

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  • Recombinant expression plasmid based on T7 promoter, and transformant and application thereof
  • Recombinant expression plasmid based on T7 promoter, and transformant and application thereof
  • Recombinant expression plasmid based on T7 promoter, and transformant and application thereof

Examples

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Embodiment 1

[0099] 1.1 Vector construction

[0100] Using the plasmid pBR322 (replicon pMB1) as a template, the primer pair 3-2F (sequence shown in SEQ ID NO: 15) and 3-2R (sequence shown in SEQ ID NO: 16) amplify the corresponding sequence on the plasmid, The PCR product was digested with KpnI and HindIII; using the plasmid pKD46 as a template, the primer pair 3-3F (sequence shown in SEQ ID NO: 17) and 3-3R (sequence shown in SEQ ID NO: 18) amplified the plasmid The fragment from the araC gene on the template to the ParaB promoter region was digested with KpnI and HindIII, and the digested product was ligated with the digested product of pBR322PCR to obtain plasmid pBR3-1.

[0101] Using the genome of Escherichia coli BL21 (DE3) as a template, primers 3-4F (sequence shown in SEQ ID NO: 19) and 3-4R (sequence shown in SEQ ID NO: 20) amplify T7 RNA polymerase, PCR The product was digested with HindIII and SalI and recovered, and the plasmid pBR3-1 was digested and recovered with the same ...

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Abstract

The invention relates to a recombinant expression plasmid, especially to a recombinant expression plasmid based on a T7 promoter. The invention also relates to a transformant containing the recombinant expression plasmid, and application of the transformant, particularly application of the transformant to fermentation production of lysine decarboxylase and the like and production of 1,5-pentamethylene diamine. The recombination expression plasmid comprises a plasmid skeleton; a replicon; a target gene and the T7 promoter controlling the expression of the target gene; and a T7 RNA polymerase gene and a sugar-induced stringent promoter controlling the expression of the T7 RNA polymerase gene, e.g., an Arabinose promoter. The transformant contains the recombinant expression plasmid as described above. The invention provides a method for fermentation production of polypeptide. The method comprises a step of culturing any transformant as described in the specification. According to the invention, cost for current induced recombinant expression inducers is lowered.

Description

technical field [0001] The present invention relates to a recombinant expression plasmid, in particular to a recombinant expression plasmid based on a T7 promoter. The present invention also relates to a transformant comprising the recombinant expression plasmid. The present invention also relates to the application of the transformant, especially in fermentation production The application of polypeptides such as lysine decarboxylase, and the further production of 1,5-pentanediamine. Background technique [0002] The present invention relates to the composition of a nucleic acid sequence and the massive expression of enzymes, proteins or polypeptides (collectively referred to as polypeptides) using the sequence. In the research of enzymes, proteins or polypeptides, most of them involve the structure and function of proteins / enzymes / polypeptides, such as the three-dimensional structure of proteins or enzymes, the catalytic performance of enzymes and the application of enzymes...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/74C12N1/21C12N9/88C12P13/00
CPCC12N9/88C12N15/70C12N15/74C12P13/001C12Y401/01018C12N2830/34
Inventor 陆文强周豪宏刘修才
Owner CATHAY R&D CENT CO LTD
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