Method of inclusion body protein renaturation and purification at the same time

Abstract

A process for renaturating and purifying the inclusion body protein includes such steps as dissolving the inclusion body protein in acidic / alkaline flowing phase or other solution, loading it in the ion-exchange chromatographic column, reversible adsorbing it by the chromatographic medium, and modifier concentration gradient elution. Its advantages are high concentration and activity of recovered protein and high purity.

Description

field of invention [0001] The invention belongs to the field of biotechnology, in particular to a method for refolding and simultaneously purifying inclusion body proteins. Background technique [0002] The development of biotechnology has made it possible to produce target proteins on a large scale. Some proteins that are rare and difficult to extract can be efficiently expressed in host cells through transgenic technology, and the most commonly used host cells are Escherichia coli. The expression of target protein in Escherichia coli has the advantages of rapidity, large quantity and low cost. However, the massive expression of foreign proteins in Escherichia coli often forms protein aggregates, namely inclusion bodies. Inclusion bodies are aggregates of randomly stretched peptide chains, without biological activity, and inclusion body proteins need to be dissolved and then refolded. [0003] The renaturation of protein is a difficult and hot...

AI Technical Summary

Problems solved by technology

The disadvantages of the general renaturation method are: mainly the low renaturation rate at low concentration, so that the whole process requires a large amount of buffer solution, a large volume of containers, and a large amount of operating time, which greatly reduces the production efficiency and increases the production efficiency. Cost of production

Dilution method is to directly dilute the inclusion body protein with refolding buffer to achieve the purpose of refolding. There are long refolding time, large amount of buffer, and large container volume, which are not suitable for large-scale production; and The refolding method of dialysis is to put the inclusion body protein in the dialysis bag, and gradually dialyze the deformation agent in the bag with the constantly changing refolding buffer to achieve the purpose of refolding. The disadvantage is that it is easy to cause protein and dialysis membrane The adsorption between the two methods, and because the refolding buffer needs to be replaced frequently, the operation steps and operation time are increased; both methods have a large buffer volume, which not only brings inconvenience to the subsequent purification process, but also increases the use of the buffer The production cost is increased, and the processing capacity of the equipment must be increased, which is not suitable for industrial production;

Washing the refolded protein with a buffer that does not contain a denaturant not only reduces the yield of refolding, but also some target proteins are adsorbed on the chromatographic medium and are not eluted from the column, resulting in a low quality recovery of the protein, and Contaminated column

Some people use this process to refold and denature the natural lysozyme protein, and the mass yield of the protein is only about 10%.

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