High cell density cultivation of recombinant E. coli for hirudin variant 1 production by temperature shift controlled by pUC18-based replicative origin.
The copy number of a plasmid, pUC-based vector, was previously shown to be affected by culture temperature. In this study, intracellular hirudin variant 1 (f-HV1) fused to porcine adenylate kinase protein was produced using recombinant Escherichia coli by temperature shift cultivation coupled with a high cell density cultivation technique for E. coli JM109. The optimal temperature for cellular growth suppressing f-HV1 production was 33 degrees C, resulting in a final dried cell concentration of 45.7 g/l, with a specific growth rate of 0.54 1/h. Optimizing the temperature-shift conditions (temperature shifted to an OD660 nm of 15 from 33 degrees C to 37 degrees C) resulted in the production of f-HV1 up to 4763 mg/l as an inclusion body with dried cell concentration of 44 g/l in 18 h.
Matsui
Toru
T
Center of Molecular Biosciences, University of the Ryukyus, Nishihara-cho, Okinawa, 903-0213, Japan. tmatsui@comb.u-ryukyu.ac.jp
Sato
Hiroaki
H
Yamamuro
Hotaka
H
Shinzato
Naoya
N
Matsuda
Hitoshi
H
Misawa
Satoru
S
Sato
Seigo
S
D000818:Animals; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D005285:Fermentation; D005818:Genetic Engineering; D006629:Hirudins / Q000235:genetics / Q000378:metabolism*; D047709:Hirudo medicinalis / Q000378:metabolism; D007218:Industrial Microbiology; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D003470:Culture Media; D006629:Hirudins; D011993:Recombinant Fusion Proteins; C072298:hirudin HV1