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Applied and Environmental Microbiology, September 2000, p. 3960-3965, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

Yoichi Kurokawa, Hideki Yanagi, and Takashi Yura^*

HSP Research Institute, Kyoto Research Park, Kyoto 600-8813, Japan

Received 20 March 2000/Accepted 5 July 2000

Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli.

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^* Corresponding author. Mailing address: 12 Hazama-cho, Shugakuin, Sakyo-ku, Kyoto 606-8071, Japan. Phone and fax: 81-75-781-7828. E-mail: tayura{at}ip.media.kyoto-u.ac.jp.

Present address: Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan.

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Applied and Environmental Microbiology, September 2000, p. 3960-3965, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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