Method for large-scale purification of recombinant human apolipoprotein Apoa-I

Abstract

Belonging to the technical field of genetic engineering recombinant protein purification, the invention relates to a method for purification of recombinant protein. The method includes: 1) centrifuging a fermentation broth to obtain a supernatant, and performing ultrafiltration and concentration on the supernatant; 2) subjecting the ultrafiltered concentrated solution to gel filtration chromatography to remove pigment and salt; 3) conducting anion chromatography on the obtained liquid, performing high salt gradient elution, and collecting a target protein peak; and 4) subjecting the collected liquid to hydrophobic chromatography to obtain pure recombinant human apolipoprotein ApoA-I. The method provided by the invention is employed to purify recombinant human apolipoprotein Apoa-I, the fermentation supernatant is subjected to ultrafiltration and concentration, further gel chromatography and hydrophobic chromatography are adopted, the final eluent is water, and can be directly subjected to freeze drying without desalination, the obtained recombinant human ApoA-I has purity of more than 96%, the recovery rate is higher than 82%, and the treatment capacity for each time is 3L. The method has the advantages of simplicity and convenience, stable process and easy amplification, can solve the scale problem in industrialization production of recombinant human apolipoprotein ApoA-I, and is suitable for separation and purification of production.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering recombinant protein purification, and relates to a method for large-scale purification of recombinant human apolipoprotein Apoa-I, in particular to a method for large-scale purification of recombinant human apolipoprotein Apoa-I based on gel chromatography and hydrophobic chromatography technology. I method. [0002] Background technique: [0003] The prior art discloses that apolipoprotein A-I (Apolipoprotein A-I, referred to as ApoA-I) is the main apolipoprotein constituting high-density lipoprotein (HDL), and studies have shown that HDL can accept cholesterol flowing out from extrahepatic tissue cells and bring it back The liver, through its ApoA-I and liver cell surface receptor recognition pathway, releases cholesteryl ester into the liver cells, and realizes the reverse transport of cholesterol from extrahepatic cells back to the liver. ApoA-I accounts for about 70% of the total ...

AI Technical Summary

Problems solved by technology

The research group of this application has successfully highly expressed recombinant ApoAI using the Pichia pastoris expression system before. The purification method used is to first obtain the crude product by cold acetone-isoelectric point fractional precipitation, and then apply Q-Sepharose ion exchange column chromatography , the recombinant human ApoAI with a purity of 95% can be obtained; but there are obvious defects in this method. Although it seems that there are few steps, the step of cold acetone is more cumbersome. Because ApoAI is secreted and expressed, the volume of the fermentation broth is large. A large amount of acetone is required, and precipitation requires low-temperature high-speed centrifugation, which consumes a lot of energy. Therefore, this method is not conducive to large-scale purification

Images