Guanidine Hydrochloride——Protein refolding, solubilizing protein

        In the process of protein renaturation, the inclusion body protein must first be dissolved. Inclusion body protein refers to the aggregation of proteins expressed by bacteria in cells to form inactive solid particles, which are amorphous and water-insoluble, and only dissolve in denaturants such as urea and guanidine hydrochloride.

        Afterwards, the denaturing agent is slowly removed to restore the target protein from the denatured fully stretched state to the normal folded structure, while the reducing agent is removed to allow the normal formation of disulfide bonds. Generally, the renaturation process of guanidine hydrochloride starts at 4M and ends at 1.5M. There are several different methods:

        Dilution and refolding: directly add water or buffer to dilute the solution and leave it overnight to make the protein precipitate out. The disadvantage is that the volume increases greatly, the denaturant dilution speed is too fast, and it is difficult to control; dialysis or ultrafiltration refolding: both denaturants Small molecular substances can gradually penetrate the porous membrane during dialysis or ultrafiltration, so that the denaturant concentration of the denatured protein solution is gradually reduced, and finally removed, so that the denatured protein is folded and refolded. The advantage is that it does not increase the volume. The removal rate of denaturant is controlled by gradually reducing the concentration of the external permeate, but the process is slow and time-consuming; on-column renaturation: The renaturation is achieved in the chromatographic process, which has been studied and successfully recently. A refolding method used in production. The advantage is that the adsorption capacity of the chromatographic fixation is relatively low compared to the denatured protein, which can improve the quality of refolding and the activity yield; at the same time, it can also separate the target protein and the impurity protein to achieve the purpose of purification; and it is convenient to recover the denaturant.

        Protein is a biological macromolecule related to life, an indispensable substance to maintain the normal progress of life, and it is also the second-generation code of life inheritance. Various researches on protein are also subjects of more interest to people, and the research on its structure is also very important for the advancement of medicine. The denaturation or renaturation of protein is one of the core issues in life science research. Among them, the denaturant guanidine hydrochloride can not only denature the protein, facilitate the study of its structure, but also dissolve the inclusion body protein during the renaturation process. Played an essential role in the research.