title: Two-step transpeptidation of the insulin precursor expressed in Pichia pastoris to insulin ester via trypsin-catalyzed cleavage and coupling.
abstract: Insulin precursor fusion protein expressed in Pichia pastoris is a single-chain protein with a spacer peptide (EEAEAEAEPK) localized at its N-terminal. Currently, the one-step transpeptidation reaction with low yield and high cost is generally employed to convert the insulin precursor fusion protein into human insulin ester. In this study, a two-step transpeptidation reaction was proposed separating the cleavage step from the coupling step so that each reaction was performed under its optimal conditions. Using this method, the total efficiency doubled and the reaction time was shortened compared with the one-step method. In addition, the amount of O-t-butyl-l-threonine t-butyl ester and trypsin dosages were reduced by 50% and 75%, respectively. This two-step transpeptidation strategy was simple and efficient and could be used for the pharmaceutical production of human insulin.
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.|Shandong Dong-e E-Jiao Co., Ltd., Shandong, People's Republic of China.|Shandong Ehua Biopharmaceutical Co., Ltd., Shandong, People's Republic of China.
authors: Haifeng
authors: H
authors: Liu
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
authors: Xiangshan
authors: X
authors: Zhou
authors: Shandong Dong-e E-Jiao Co., Ltd., Shandong, People's Republic of China.
authors: Shousheng
authors: S
authors: Tian
authors: Shandong Dong-e E-Jiao Co., Ltd., Shandong, People's Republic of China.
authors: Xianghui
authors: X
authors: Hao
authors: Shandong Dong-e E-Jiao Co., Ltd., Shandong, People's Republic of China.|Shandong Ehua Biopharmaceutical Co., Ltd., Shandong, People's Republic of China.
authors: Jinhua
authors: J
authors: You
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
authors: Yuanxing
authors: Y
authors: Zhang
mesh_terms: D055162:Biocatalysis; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D007328:Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D004952:Esters; D007328:Insulin; D014357:Trypsin
keywords: Insulin; Pichia pastoris; cleavage; coupling; insulin precursors; transpeptidation