Production of active bovine cathepsin C (dipeptidyl aminopeptidase I) in the methylotrophic yeast Candida boidinii.
The heterologous production of active bovine cathepsin C (CTC; dipeptidyl aminopeptidase I) was investigated. Attempts to express CTC in Escherichia coli were hampered by formation of inclusion bodies that were partially degraded. To overcome this impediment, secretion of recombinant CTC was attempted in the methylotrophic yeast Candida boidinii. A DNA fragment encoding bovine procathepsin C was synthesized based on preferred codon usage in C. boidinii and placed downstream of the C. boidinii proteinase A signal sequence resulting in secretion of active CTC into the culture medium. The gene was expressed under the control of the methanol-inducible formate dehydrogenase gene promoter. Production levels were significantly improved by using a protease-deficient strain, changing medium composition, and by lowering the temperature of induction. When the recombinant C. boidinii was grown for 90 h in a jar-fermenter, active CTC was secreted with a yield of up to approximately 12 mg/l.
Komeda
T
T
Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa, Japan. t-komeda@kirin.co.jp
Tazumi
K
K
Shimada
H
H
Kano
K
K
Hayashi
T
T
Saito
H
H
Tsumura
H
H
Kato
N
N
Sakai
Y
Y
Kondo
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002175:Candida / Q000378:metabolism*; D020789:Cathepsin C / Q000096:biosynthesis*; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins; D020789:Cathepsin C