title: A novel approach for secretion of heterologous proteins with correct n-terminal processing by using α-factor pre sequence in Pichia pastoris.
abstract: Numerous proteins have been secreted in P. pastoris by fusing the target gene with α-factor pre-pro sequence at Kex2 endopeptidase cleavage site. However, in some instances the product cannot be correctly processed due to aberrant cleavage by Kex2 endopeptidase such as aprotinin. In this study, an aprotinin gene was cloned into pPIC9K at the signal peptidase cleavage site through a single NheI restriction site designed at the 3'end of the α-factor signal sequence preregion, and transformed into GS115 host cell. By G418 resistance and ELISA assay, a high-yield recombinant was selected. After fed-batch cultivation in a 7-L bioreactor, the product was efficiently secreted into culture medium and accumulated up to ~4.7 mg L⁻¹. MALDI-TOF/MS and N-terminal analyses confirmed its authenticity. Thus, a novel cloning strategy for secretion of aprotinin with correct N-terminal processing in P. pastoris has been developed which can be potentially applied to other proteins.
authors.affiliation: State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, People's Republic of China.
authors.forename: Lu
authors.initials: L
authors.lastname: Shen
authors.forename: Zhenyun
authors.initials: Z
authors.lastname: Liu
authors.forename: Fenli
authors.initials: F
authors.lastname: Xie
authors.forename: Xuerui
authors.initials: X
authors.lastname: Zhang
authors.forename: Shuhua
authors.initials: S
authors.lastname: Tan
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D007611:Aprotinin