High level expression, purification and activation of human dipeptidyl peptidase I from mammalian cells.
Dipeptidyl peptidase I (DPPI) plays a crucial role in maturation of many regulatory peptides and has been suggested as a pharmaceutical target in several inflammatory diseases. It is also a useful processing enzyme for the generation of authentic protein products by catalyzing the removal of N-terminal fusion peptides. We used a robust transient transfection system in human embryonic kidney 293 cells to exploit expression and activation of DPPI from chicken, rat and man for the development of an industrial production process. The expression of human and rat DPPI was significantly higher in the human HEK293 cell line than that obtained with avian DPPI. A CHO K1SV stable cell line was selected as the optimal stable host system for production of human DPPI yielding expression levels higher than 1.5 g/L. The secreted pro-DPPI underwent auto-maturation during defined buffer conditions during the purification steps. Active human DPPI was purified with a three-step purification strategy employing: Butyl Sepharose 4 Fast Flow, Sephadex G-25 Medium and Q Sepharose Fast Flow chromatography. The final yield of active enzyme was approximately 1 g/L cell culture. The enzyme exhibited exopeptidase activity against both a dipeptide-p-nitroanilide substrate and N-terminally extended MEAE-hGH (Met-Glu-Ala-Glu-human growth hormone). In conclusion, an efficient production process for recombinant human DPPI has been developed including a highly efficient and stable CHO cell system and an efficient purification procedure, which is simple and easy to scale for industrial purposes. The present data facilitates not only industrial applications of DPPI as a processing enzyme, but also provides active enzyme useful in the identification of small molecule inhibitors.
Yang
Wei
W
Beijing Novo Nordisk Pharmaceuticals Sci & Tech Co. Ltd., Beijing 102206, China. weyg@novonordisk.com
Xia
Wenjuan
W
Mao
Jingjing
J
Xu
Daqi
D
Chen
Jianhe
J
Feng
Shan
S
Wang
Jianhua
J
Li
Hua
H
Theisen
Claus Friis
CF
Petersen
Jørn Meidahl
JM
Thórólfsson
Matthías
M
Rasmussen
Hanne Benedicte
HB
Junker
Flemming
F
Boel
Esper
E
Su
Jing
J
D000818:Animals; D016466:CHO Cells; D020789:Cathepsin C / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D002863:Chromogenic Compounds; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D002863:Chromogenic Compounds; D017077:Culture Media, Conditioned; D011993:Recombinant Fusion Proteins; D020789:Cathepsin C