title: Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris.
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.
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China.
authors.forename: Yugang
authors.initials: Y
authors.lastname: Guo
authors.forename: Gan
authors.initials: G
authors.lastname: Luan
authors.forename: Guodong
authors.initials: G
authors.lastname: Shen
authors.forename: Lidan
authors.initials: L
authors.lastname: Wu
authors.forename: Hao
authors.initials: H
authors.lastname: Jia
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Zhong
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.forename: Guangwei
authors.initials: G
authors.lastname: Li
authors.forename: Yi
authors.initials: Y
authors.lastname: Shen
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.forename: Siyi
authors.initials: S
authors.lastname: Hu
authors.forename: Weihua
authors.initials: W
authors.lastname: Xiao
mesh_terms: D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D061249:Batch Cell Culture Techniques / Q000379:methods*; D019149:Bioreactors / Q000382:microbiology; D005285:Fermentation; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; C075005:GNLY protein, human; D011994:Recombinant Proteins