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Protein modification method for increasing yield of interleukin 1 receptor related kinase proteins

An interleukin and kinase protein technology, which is applied in the field of protein transformation to increase the production of interleukin 1 receptor-related kinase proteins, can solve the problems of heavy social burden, the patient's condition cannot be effectively controlled, and the like, so as to improve the expression yield Effect

Inactive Publication Date: 2020-03-24
武汉赛维尔生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the treatment of these diseases is still facing a severe test, and the condition of many patients cannot be effectively controlled, causing a heavy burden on the patient's family and society.

Method used

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  • Protein modification method for increasing yield of interleukin 1 receptor related kinase proteins
  • Protein modification method for increasing yield of interleukin 1 receptor related kinase proteins
  • Protein modification method for increasing yield of interleukin 1 receptor related kinase proteins

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Embodiment 1

[0022] When the mouse IRAK1 protein was expressed in the prokaryotic expression system, it was found that the molecular weight of the protein expressed was significantly different from the molecular weight of the protein in the selected region. The check sequencing result completely matched the original sequence and could be translated normally. Analysis of the reason found that there is a secondary translation initiation site in the gene sequence, that is, a sequence similar to the ribosome binding site (AAGGAGG) plus an appropriate spacer sequence (usually 5 to 13 nucleotides), located in AUG (Met) Upstream of the codon, the presence of this secondary translation initiation site may cause the expressed protein to be truncated and affect the normal expression of the protein.

[0023] The present invention designs two pairs of primers (F1 / R1, Fm / Rm) to synonymously mutate the similar ribosome binding site in the gene sequence through the overlap PCR technology, and the base at the...

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Abstract

The invention belongs to the technical field of gene technologies and microorganisms, and particularly relates to a protein modification method for increasing the yield of interleukin 1 receptor related kinase proteins. The protein modification method comprises the following steps: cDNA prepared by reverse recording of RNA extracted from mouse liver tissue or lung tissue is used as a template, twopairs of primers F1 / R1 and Fm / Rm are adopted, synonymous mutation on base sequences 'CAGGAG' from the 115th site to the 120th site in an original gene sequence into a base sequence 'CAAGAA' by virtueof an Overlap PCR amplification technology is carried out; and the mutated gene segment is constructed into a pET 32a expression vector, converting into escherichia coli BL21 (DE3) for protein induced expression is carried out, and purifying is carried out to obtain the interleukin 1 receptor related kinase proteins. According to the method, normal expression of the interleukin 1 receptor relatedkinase proteins is achieved, and the expression yield of the proteins is greatly increased.

Description

Technical field [0001] The invention belongs to the field of gene technology and microbial technology, and specifically relates to a protein modification method for improving the production of interleukin 1 receptor related kinase protein. Background technique [0002] Interleukin-1 receptor-associated kinase (Interleukin-1 Receptor-Associated Kinase, IRAK1) is a type of intracellular kinase, in the signal pathway mediated by Toll-like receptor (TLR) and interleukin-1 receptor (IL1-R) It plays an important role in regulating the production of pro-inflammatory factors, and plays a positive or negative role in regulating inflammation. [0003] Recent studies have found that inflammation is closely related to the occurrence of many diseases. In neurological diseases, the pathophysiological process of cerebrovascular diseases, neurodegenerative diseases such as AD, epilepsy, etc. all involve inflammation. At present, the treatment of these diseases is still facing severe tests. Many p...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/10C12N15/70
CPCC12N9/1205C12N15/1031C12N15/70C12Y207/10002C12Q2531/113
Inventor 刘伟林刘姗姗李千张高英赵红洲
Owner 武汉赛维尔生物科技有限公司
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