Expression of aqualysin I (a thermophilic protease) in soluble form in Escherichia coli under a bacteriophage T7 promoter.
The thermophilic protease aqualysin I (AQI) gene (aquI), derived from Thermus aquaticus YT-1, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid. The plasmid was introduced into two strains of E. coli JM109 (DE3), one carrying and one lacking an F' episome, which carries the lacIq gene. Upon cultivation the strain carrying an F' episome produced AQI as an insoluble fusion protein (74kDa) with the T7 gene 10 protein. This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity. On the other hand, when the strain lacking an F' episome was used as a host cell for aquI expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form. This soluble protein could be processed into active AQI by heat treatment. Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction.
Sakamoto
S
S
Life Science Research Laboratory, Japan Tobacco Inc., Yokohama.
Terada
I
I
Iijima
M
M
Ohta
T
T
Matsuzawa
H
H
D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D012697:Serine Endopeptidases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012995:Solubility; D013824:Thermus / Q000201:enzymology*
D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D012697:Serine Endopeptidases; C055102:aqualysin I