Renaturing and purifying method of mycobacterium-tuberculosis recombinant inclusion-body protein

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a renaturing and purifying method of mycobacterium-tuberculosis recombinant inclusion-body protein, which comprises the following steps of: preparing preliminarily-purified rTPA38KDa inclusion-body protein, cracking the rTPA38KDa inclusion-body protein, renaturing and purifying by a primary anion-exchanging chromatographic method, further purifying by a secondary anion-exchanging chromatographic method, and ultrafiltering to obtain a stock solution of the rTPA38KDa inclusion-body protein. In the invention, the renaturation and the purification of target protein are finished through two times of anion-exchanging chromatography by adopting a method of firstly renaturing and then purifying, the total yield of the target protein reaches more than 30 percent, and the purity reaches more than 95 percent; and compared with the prior art, the method adopted by the invention can be used for industrial amplification, is convenient for quality control and has fewer process steps, lower production cost and higher efficiency.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a method for renaturation and purification of mycobacterium tuberculosis recombinant inclusion body protein. Background technique [0002] Tuberculosis (TB) is a chronic zoonotic infectious disease caused by Mycobacterium tuberculosis (MTB), which can affect various tissues and organs throughout the body, among which pulmonary tuberculosis is the most common. Due to the emergence of MTB drug-resistant strains, AIDS combined with MTB infection and the flow of high-risk groups of tuberculosis around the world, the global epidemic of TB has been caused. At present, tuberculosis has become one of the main diseases that cause adult deaths from infectious diseases all over the world. According to the report of the World Health Organization, nearly 1 / 3 of the people in the world have been infected with MTB. There are about 20 million active tuberculosis patients in the world,...

AI Technical Summary

Problems solved by technology

Among them, 1) Dilution renaturation is a traditional renaturation method, mainly to directly reduce the concentration of denaturant through dilution, and control the protein concentration to 10-100 μg / ml or lower to reduce the coagulation effect. : Industrial use of this kind of refolding method requires a lot of containers and a large amount of refolding buffer. The protein concentration is difficult to control, and it is difficult to recover the refolded protein; Refolding also cannot solve the problem of coagulation, and is not suitable for industrial production due to the need for multiple replacement buffers and the lack of suitable industrial-scale dialysis devices. This method can only be used on a laboratory scale; 3) ultrafiltration refolding The method is similar to dialysis renaturation. It mainly reduces the concentration of denaturant through the ultrafiltration device. The process is faster than dialysis and easy to scale up. However, it is difficult to solve the problem of coagulation like dilution renaturation and dialysis renaturation.

Among them, the column chromatography method usually requires 3-4 steps of column chromatography, and its disadvantages are: many process steps, low efficiency, and difficult quality control; while the method of affinity chromatography is mainly to fuse and express the target protein with the His6 tag sequence , and then purified by metal chelate chromatography

Although the purification efficiency of this method is high, its disadvantages are: 1) It is necessary to introduce a heterologous His6 sequence at the end of the target protein, so the purified product cannot be applied to the human body; 2) the affinity chromatography medium has a short service life and is expensive. Not suitable for large-scale industrial production

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