Minimum promoter for enhancing activity and expression of exogenous protein in bacteria

A technology of exogenous proteins and promoters, applied in biochemical equipment and methods, microbial assays/tests, resistance to vector-borne diseases, etc. Problems such as being detected to achieve the effect of enhancing expression and activity

Active Publication Date: 2021-10-26
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Target protein cannot be detected or is detected by sensitive techniques such as Western blot
At very low levels (less than micrograms per liter of culture), the problem is usually either that the heterologous protein is acting deleteriously or that the protein itself is not stable
[0005] (2) Protein inactivation
The protein may still be of poor quality, i.e. it is not as active as it should be

Method used

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  • Minimum promoter for enhancing activity and expression of exogenous protein in bacteria
  • Minimum promoter for enhancing activity and expression of exogenous protein in bacteria
  • Minimum promoter for enhancing activity and expression of exogenous protein in bacteria

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Experimental program
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Embodiment

[0025] A minimal promoter that enhances the activity and expression of foreign proteins in bacteria is 265 nucleotides upstream of the coding region of the SspH1 gene of Salmonella enterica subsp. 1.

[0026] Identification of a minimal promoter that enhances expression of foreign proteins in bacteria:

[0027] 1. Clone the SspH1 gene promoter of Salmonella enterica subspecies Chlamydia enterica typhimurium strain RM13672;

[0028] Primers were designed according to the sequence 615 nucleotides in length upstream of the SspH1 coding region in the chromosomal sequence of Salmonella enterica subsp. Use the primer pair pSspH1-615-F / pSspH1-R1, bacterial genomic DNA and high-fidelity Tag DNA polymerase to establish a polymerase chain reaction (PCR). The reaction conditions are: 95°C for 2min, then 35 cycles of 95°C for 30sec , 55°C 30sec, 72°C 1min; finally 72°C 5min. The 0.6 kb DNA band generated by PCR was purified by agarose gel electrophoresis.

[0029] The primer sequence...

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Abstract

The invention belongs to the technical field of biology, and discloses a minimum promoter for enhancing activity and expression of exogenous protein in bacteria. According to the minimum promoter, the minimum promoter capable of enhancing the activity and the expression of the exogenous protein in the bacteria is screened in a deletion mutation manner, and the minimum promoter is 265 nucleotides on the upstream of a gene coding region of salmonella enteric subspecies enteric chlamydia mouse typhimurium strain RM13672SspH1, and the nucleotide sequence of the minimum promoter is represented by SEQ.ID.NO: 1. The obtained minimum promoter for enhancing the activity and expression of the exogenous protein in the bacteria still keeps activity in escherichia coli, is a promoter with general activity, has the potential to be applied to other different types of prokaryotic bacteria, and can be used as a tool for improving the protein yield and the protein activity in the industry or scientific research, and scientific research, biological medicine industrial production and preparation of anti-infectious disease vaccines are facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a minimum promoter for enhancing the activity and expression of foreign protein in bacteria. Background technique [0002] In scientific research and biomedical industry production and anti-infectious disease vaccine preparation, it is often necessary to express or purify biologically active bacteria in prokaryotic bacteria (including but not limited to Escherichia coli, Salmonella, Shigella, Mycobacterium tuberculosis, etc.) protein. [0003] However, the expression of foreign proteins in bacteria often encounters the following problems: [0004] (1) No output or low output. The protein of interest cannot be detected or is detected by sensitive techniques such as Western blot. At very low levels (less than micrograms per liter of culture), the problem is usually either that the heterologous protein is acting deleteriously or that the protein itself is not stable. [0005] (2) Pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/70C12Q1/6897
CPCC07K14/255C12N15/10C12N15/70C12Q1/6897C12Q2531/113Y02A50/30
Inventor 邢力潘元晴吴长新
Owner SHANXI UNIV
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