Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris
Yugang Guo, Gan Luan, Guodong Shen, Lidan Wu, Hao Jia, Yongjun Zhong, Rui Li, Guangwei Li, Yi Shen, Jie Sun,Siyi Hu, Weihua Xiao
Abstract
Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.