| Summary: | 碩士 === 明志科技大學 === 化學工程系生化工程碩士班 === 107 === The main host for over-expression of recombinant proteins in prokaryotic system is Escherichia coli. However, one of the major obstacles in E. coli is the recombinant in inclusion body formation. Then the target is an insoluble form. The strong protein denaturant such as 8 M urea or 6 M guanidinium hydrochloride is applied to denature the structure of protein for solving in an aqueous solution. Finally, urea or guanidinium hydrochloride is removed gradually by dialysis to allow refolding of the denatured proteins.
The chitinase, AsChi61, possess high chitolytic activity and SDS resistance. This indicates its structure is too rigid to tolerate the environmental factors when applied in industry. In addition, the major products obtained from chitin digested by AsChi61 are N-acetamidine hexahexose. Hexameric oligochitin was known to be immune enhancer with anti-tumor activity. The reAsChi61 with those advantages are expressed in inclusion body in Escherichia coli system.
In this study, reAsChi61 was expressed in the pET15b system, and then analyzed on SDS-PAGE electrophoresis. In the purification, urea was applied to solve the protein. With different concentration of urea, some inclusion bodies were denatured. However, the His-tag of reAsChi61 was still bound to the resin. After elution, the expression activity and amount of the recombinant chitinase can be improved.
|
|---|
