A kind of refolding method of recombinant protein

Abstract

The invention relates to a recombinant protein renaturation buffer solution as well as a preparation method and an application thereof. The buffer solution consists of a basis solution, 0.5% of beta-mercaptoethanol, 1% of tween-20, 10mM of beta-cyclodextrin, 1M of L-cysteine and 4M of urea. The preparation method comprises the following steps: firstly, adding 4M of urea into the basis solution, secondly, adding 0.5% of beta-mercaptoethanol, 1% of tween-20, 10mM of beta-cyclodextrin and 1M of L-cysteine according to volume ratio, so as to form the recombinant protein renaturation buffer solution. The buffer solution provided by the invention is applied to recombinant protein renaturation with the primary protein concentration greater than 1mg / mL, is simple to operate, low in cost and convenient in industrial production, the volume after renaturation is slightly increased, the protein concentration is slightly diluted, and the renaturation rate is high.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a recombinant protein refolding buffer and its preparation method and application. Background technique [0002] The incomplete modification, misfolding and aggregation of the peptide chain due to the expression of the recombinant protein in bacteria generally exists in the form of inclusion bodies. Break the bacteria to obtain pure inclusion bodies. After being dissolved by denaturant (6M guanidine hydrochloride or 8M urea), the inclusion body must be refolded to make the protein fold correctly, improve solubility and restore biological activity before it can reach the level of clinical application. If the protein cannot be renatured well, it will directly manifest as a decrease in solubility and protein precipitation and precipitation, resulting in a large loss of protein. Due to the limitation of technical level, the renaturation and purification of protein is the bottleneck that ...

AI Technical Summary

Problems solved by technology

There are various problems in the above-mentioned renaturation methods. For example, dialysis renaturation puts the denatured protein into the dialysis bag, and realizes protein renaturation by slowly reducing the concentration of the denaturant. Although the operation is simple, it takes a long time and the processing volume is small. Unable to be applied on a large scale

Dilution renaturation is to dilute the denatured protein solution with water or renaturation buffer to slowly decrease the concentration of denaturant and achieve the purpose of protein renaturation. However, there is no general renaturation buffer at present, and according to current literature reports, by When this method is refolded, the protein concentration needs to be controlled at a low level, which is not suitable for high-concentration proteins. In the current dilution refolding method, the initial protein concentration is generally controlled at 10-100 μg / mL, or even lower

Therefore, the traditional refolding method will dilute the protein in a large amount, which will eventually cause a rapid increase in volume, making it difficult to concentrate the protein in the later stage, and it is not suitable for industrial production.

The renaturation of artificial molecular chaperones is commonly used for Hsp family proteins and GroES / GroEL, etc. Artificial molecular chaperones can significantly increase the protein renaturation rate, but at the same time it will also significantly increase the production cost

Column chromatography renaturation and reverse micelle dissolution renaturation require special materials and equipment, high cost, not suitable for large-scale preparation in the pharmaceutical industry, etc., and the operation process is relatively cumbersome

In conclusion, there is no report on renaturation for initial protein concentration higher than 1 mg / mL.

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