Method for preparing polypeptide on basis of escherichia coli expression system

Abstract

The invention discloses a method for preparing polypeptide on the basis of an escherichia coli expression system. According to the method, firstly, a target gene of the polypeptide is recombined intoa vector plasmid, the recombined plasmid is transformed into escherichia coli DH5 alpha and amplified, screening is performed through resistance genes, sequencing verification is performed, escherichia coli DH5 alpha into which the recombined plasmid is successfully transformed is obtained, the recombined plasmid is extracted with an alkaline lysis method and transformed into an escherichia coli BL21(DE3)Star pLysS strain, expression of a target polypeptide is induced, thalli are cultured and collected, finally, the thalli are resuspended through a PBS buffer solution, crushed and centrifuged,precipitates are taken, the resuspension and centrifugation steps are repeated by use of a Triton X-100-containing buffer solution, precipitates are repeatedly cleaned, then, dissolution and precipitation are performed, and the target polypeptide is obtained. By use of an inclusion body formed by escherichia coli BL21(DE3)Star through high expression, the polypeptide purification step is simplified while the yield of the polypeptide is greatly increased, the purity of the polypeptides is increased, and the method has application prospects in the field of preparation of the polypeptide throughgenetic recombination.

Description

technical field [0001] The invention relates to a method for preparing a polypeptide based on an Escherichia coli expression system, in particular to a method for preparing a polypeptide by using the Escherichia coli BL21 (DE3) Star strain and utilizing the characteristics of inclusion bodies to express and purify, and belongs to the field of biotechnology. Background technique [0002] Peptides have extremely high nutritional value and rich diversity, and are research and development hotspots in the fields of biology, medicine, and pharmacy. Bioactive peptides are not only essential nutrients for human growth and development, but also play a role in regulating human physiological functions (Li Jianrong, Feng Ping, Research Progress of Functional Peptides, Food Science, 2004, 25(11): 415-419 ). At present, the more common methods for preparing polypeptides include protease hydrolysis, chemical synthesis, gene recombination and extraction and separation. In recent years, th...

AI Technical Summary

Problems solved by technology

However, there are still some problems in using genetically engineered strains to express and prepare polypeptides. How to further increase the yield of polypeptides and simplify the steps of purifying polypeptides has always been the focus of this technical field.

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