Efficient production of active Vibrio proteolyticus aminopeptidase in Escherichia coli by co-expression with engineered vibriolysin.
The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.
Sonoda
Hiroyuki
H
Research Center, Research Division, JCR Pharmaceuticals Co, Ltd, Kobe 651-2241, Japan. sonoda-h@jcrpharm.co.jp
Daimon
Katsuya
K
Yamaji
Hideki
H
Sugimura
Atsushi
A
D000626:Aminopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D000626:Aminopeptidases; C038222:bacterial leucyl aminopeptidase; D008666:Metalloendopeptidases; C073831:vibriolysin protein, Vibrio proteolyticus