A kind of recombinant trypsin purification method

Abstract

The invention relates to the field of biotechnology, and provides a recombinant trypsin purifying method. The method comprises the steps of firstly activating the recombinant trypsinogen generated by yeast fermentation, and then purifying with macroporous adsorption resin and cation exchange chromatography to obtain pure trypsin. A process of dilution or ultrafiltration of the fermentation liquid for directly purifying trypsinogen in the yeast fermentation liquid is avoided, and separate enzyme digestion pretreatment on the zymogen is not required, thus the purifying steps are saved, the operation is simple, and the yield is relatively high; moreover, the chromatography filler is cheap, and the method is suitable for large-scale production. Experiments indicate that the purity and activity of the recombinant trypsin are both relatively high, and the recombinant trypsin can be used as a raw material for industrial production.

Description

[0001] This application claims the priority of the Chinese patent application with the application number 201210450131.0 and the invention title "A Method for Purifying Recombinant Trypsin" submitted to the China Patent Office on November 12, 2012, the entire contents of which are incorporated in this application by reference . technical field [0002] The invention relates to the field of biotechnology, in particular to a method for purifying recombinant trypsin. Background technique [0003] Trypsin (Trypsin, EC3.4.21.4) is a serine protease that can specifically cleave basic amino acids, such as the carboxy-terminal peptide bonds of lysine and arginine. In vertebrates, acts as a digestive enzyme. Its precursor, trypsinogen, is synthesized in the pancreas and secreted as a component of pancreatic juice. After activation by enterokinase or trypsin, it becomes active trypsin. Trypsin can also limit and decompose the precursors of other enzymes such as chymotrypsinogen, car...

AI Technical Summary

Problems solved by technology

Therefore, Pichia pastoris fermentation broth needs to be diluted or ultra-filtered to use cation exchange resins to capture the zymogen, but the method of ultra-filtration is not suitable for large-scale production, and the dilution process before purification will cause serious problems in large-scale production. A large waste of water resources, and the volume increases after dilution, resulting in prolonged purification time

[0007] Macouzet M et al. fused a histidine tag to the C-terminus of the recombinant trypsinogen, and purified the recombinant trypsinogen through the recognition of the histidine tag, which simplified the purification process and avoided the The fermentation broth is diluted or ultrafiltered, but the introduced histidine tag cannot be completely removed, and the residual histidine tag is easy to affect the later industrial application

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