Purified renaturation method of L-aspartic acid oxidase inclusion bodies

Abstract

The invention provides a purified renaturation method of L-aspartic acid oxidase inclusion bodies. The purified renaturation method comprises the following steps that precipitates of the inclusion bodies are collected, and fully washed, denatured proteins are purified by a nickel column, the purified denatured proteins are diluted with a buffer solution D until the protein concentration is lower than 1mg/ml, and placed in a dialysis buffer solution, the dialysis temperature is kept at 4 DEG C, dialysis lasts for more than 12 hours, and supernatants are centrifugally collected. Finally, the supernatants are concentrated by an ultrafiltration tube, and then monomeric proteins and agrin are purified and separated by a molecular sieve to obtain homogeneous natural active proteins. The purified renaturation method of the L-aspartic acid oxidase inclusion bodies disclosed by the invention solves the problem of L-aspartic acid oxidase renaturation, purification and renaturation on the L-aspartic acid oxidase inclusion bodies are carried out with a convenient and fast method, the inclusion bodies are further rinsed and purified by the nickel column to obtain the denatured proteins with higher purity, denaturants are removed gradually through a one-step dialysis renaturation process, the proteins are folded to be protein molecules with activity, the renaturation efficiency of the proteins is not less than10%, and then the protein molecules are further dialyzed and purified by the molecular sieve to obtain high-purity enzymes with activity.

Description

technical field [0001] The invention relates to a method for purifying and refolding L-aspartic acid oxidase inclusion bodies, which belongs to the protein expression and purification process in the field of bioengineering. Background technique [0002] In the field of protein engineering, how to obtain biologically active proteins is a key issue, which is of great significance for the study of gene functions, the development of protein therapeutics and the preparation of biological materials. Escherichia coli, the most widely used expression host, often presents proteins in the form of insoluble inclusion bodies. How to refold and obtain biologically active soluble proteins from inactive inclusion bodies has become a major problem in the field of protein engineering. [0003] When a protein is expressed in a large amount in a prokaryotic host, due to the limitation of folding factors in the host cell, such as too fast protein expression or an inappropriate protein folding p...

AI Technical Summary

Problems solved by technology

At present, a variety of L-aspartate oxidases with different sources and functions have been heterologously expressed in E. coli, but the yield is low, and most of the proteins exist in the form of inactive inclusion bodies

However, there is no effective method for renaturation of inclusion bodies, which severely restricts the industrial application of this type of enzyme.

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