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Expression and Purification of Peach Receptor-Like Protein Kinase

A technology of receptor protein and purification method, which is applied in the field of expression and purification of peach receptor-like protein kinase, can solve the problems of difficult expression, long gene, complex protein structure, etc., and achieve the effect of reducing the difficulty of expression

Active Publication Date: 2021-01-15
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Abstract
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Problems solved by technology

However, the FERONIA protein has a complex structure and a long gene, and it is difficult to express it after it is successfully constructed in the E. coli expression system

Method used

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  • Expression and Purification of Peach Receptor-Like Protein Kinase
  • Expression and Purification of Peach Receptor-Like Protein Kinase
  • Expression and Purification of Peach Receptor-Like Protein Kinase

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Embodiment

[0037] The peach receptor-like protein kinase expression and purification method comprises the following steps:

[0038] 1. Preparation of recombinant engineering strain BL21-pET-28a-FERONIA-KD containing His tag

[0039] 1.1 Through the biological analysis of the peach receptor-like protein kinase gene sequence, it is concluded that the peach receptor-like protein kinase gene sequence is divided into three parts: intracellular domain, transmembrane domain and extracellular domain. The analysis results are shown in figure 1 , and the amino acid sequence SEQ ID NO.1 of the peach receptor-like protein kinase and the nucleotide sequence SEQ ID NO.2 of the peach receptor-like protein kinase were obtained by bioanalytical methods;

[0040]1.2 Using the intracellular domain gene sequence of the peach receptor-like protein kinase as a template, Oligao 6 was used to design primers to obtain the upstream primer P1, whose nucleotide sequence is shown in SEQ ID NO.5: CGCCACCGTAGACATGGGAA...

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Abstract

The invention discloses an expression and purification method for a peach receptor-like protein kinase. The method comprises the following steps: connecting intracellular domain cDNA of the receptor-like protein kinase with pET-28a(+) to obtain pET-28a(+)-FERONIA-KD, transforming the pET-28a(+)-FERONIA-KD into escherichia coli BL21(DE3) to obtain BL21-pET-28a-FERONIA-KD, optimizing the concentration of an inducer to improve the protein expression of the BL21-pET-28a-FERONIA-KD, and finally optimizing the pH of a washing buffer solution and an elution buffer solution used in a Ni-column purification process and an imidazole concentration to obtain high-concentration BL21-pET-28a-FERONIA-KD protein. The method reduces the difficulty of expression of the peach receptor-like protein kinase, and improves the expression of the protein and the purification effect of the protein.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a peach receptor-like protein kinase expression and purification method. Background technique [0002] Peach receptor-like protein kinase (FERONIA) is an enzyme that catalyzes protein phosphorylation, and is a transmembrane protein composed of an outer malectin domain, a transmembrane domain and an inner serine / threonine kinase domain. The extracellular part of FERONIA recognizes ligands, and the intracellular part has kinase activity. It cooperates with hormones and plays an important role in the process of plant response to environmental signals. However, the FERONIA protein has a complex structure and a long gene, and it is difficult to express it after it is successfully constructed in the E. coli expression system. Contents of the invention [0003] It is an object of the present invention to solve at least the above-mentioned problems and to provide at least ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/70
CPCC12N9/12C12N15/70
Inventor 赵垚垚段玉权林琼宋丛丛齐淑宁
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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