Optimized culture conditions for the efficient production of porcine adenylate kinase in recombinant Escherichia coli.
Temperature shift cultivations with amino acid supplementation were optimized to produce porcine adenylate kinase (ADK) in recombinant Escherichia coli harboring a pUC-based recombinant plasmid under the control of the trp promoter. With regard to temperature control, the culture condition was initially maintained at 35 degrees C for cellular growth, but ADK expression was suppressed until the late logarithmic growth phase; subsequently, a temperature shift was applied (from 35 degrees C to 42 degrees C), which resulted in maximal ADK production. In addition, supplementation of amino acids, especially valine and leucine, during the temperature shift stimulated ADK expression from 3.5% to 9.2% and 8.6% of the total protein, respectively. After optimization, 1 g ADK per liter was produced within 16 h of cultivation with a dry cell weight of 21.8 g/l. In this system, there was no loss of the recombinant plasmid during cultivation without selective pressure.
Matsui
Toru
T
Center of Molecular Biosciences, University of the Ryukyus, 1 Sembaru Nishihara-cho, Okinawa 903-0213, Japan. tmatsui@comb.u-ryukyu.ac.jp
Togari
Takashi
T
Misawa
Satoru
S
Namihira
Tomoyuki
T
Shinzato
Naoya
N
Matsuda
Hitoshi
H
Sato
Seigo
S
D000263:Adenylate Kinase / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D013552:Swine; D013696:Temperature
D000596:Amino Acids; D000263:Adenylate Kinase