Comparison the effect of cytoplasmic chaperones co-expression on cytoplasmic and periplasmic production of activin A protein in E. Coli

Activin A, a member of transforming growth factor β superfamily (TGF-β), is a dimmer of two inhibin βA subunits. This protein plays many important roles in the body, such as cell growth and differentiation, maintenance and survival of the neurons, anti-inflammatory role, wound healing, and hematopoiesis; so it can be used as a therapeutic agent in the treatment of related diseases. The goal of this study is recombinant production of soluble activin A with correct structure in the cytoplasmic and periplasmic space of E. coli. For this purpose, cytoplasmic chaperones GroEL/GroES, DnaK/DnaJ and TF (Trigger factor) were expressed in order to help protein folding and prevent the formation of protein aggregates (inclusion body) as well as increase the production of soluble protein. It is worth noting that the co-expression of cytoplasmic chaperones not only affects the protein folding but also increases the production of recombinant proteins. After cytoplasmic and periplasmic expression of activin A individually and simultaneously with cytoplasmic chaperones, soluble proteins were extracted and the level of protein expression in each case was evaluated with western blotting technique and Image J software. The results showed that GroEl/GroES chaperones increased the cytoplasmic expression of soluble activin A due to assisting in protein folding; but in the periplasmic expression, TF was the best chaperone because it is the first chaperone bound to a synthesizing protein. As in the Sec secretory pathway, the protein is translocated to the periplasmic space with unfolded structure, TF can increase the amount of protein secreted from this pathway. Also, the results of structural analysis using CD spectroscopy showed the correct secondary structure of the protein produced in both cases.

کلیدواژه ها:

soluble protein ، Sec secretory pathway ، TF chaperone