TY - JOUR
T1 - Development of a long-acting Fc-fused cocaine hydrolase with improved yield of protein expression
AU - Chen, Xiabin
AU - Deng, Jing
AU - Zheng, Xirong
AU - Zhang, Jinling
AU - Zhou, Ziyuan
AU - Wei, Huimei
AU - Zhan, Chang Guo
AU - Zheng, Fang
N1 - Publisher Copyright:
© 2019 Elsevier B.V.
PY - 2019/6/1
Y1 - 2019/6/1
N2 - Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a ~2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc(M3)-(PAPAP) 2 -CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP) 2 -CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105 ± 7 h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP) 2 -CocH3 has a ~21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP) 2 -CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.
AB - Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a ~2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc(M3)-(PAPAP) 2 -CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP) 2 -CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105 ± 7 h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP) 2 -CocH3 has a ~21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP) 2 -CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.
KW - Butyrylcholinesterase
KW - Cocaine hydrolase
KW - Protein engineering
KW - Protein production
UR - http://www.scopus.com/inward/record.url?scp=85064251024&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85064251024&partnerID=8YFLogxK
U2 - 10.1016/j.cbi.2019.04.012
DO - 10.1016/j.cbi.2019.04.012
M3 - Article
C2 - 30986387
AN - SCOPUS:85064251024
SN - 0009-2797
VL - 306
SP - 89
EP - 95
JO - Chemico-Biological Interactions
JF - Chemico-Biological Interactions
ER -