This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Okuno, K. Articles by Kinoshita, S. Search for Related Content PubMed PubMed Citation Articles by Okuno, K. Articles by Kinoshita, S. Agricola Articles by Okuno, K. Articles by Kinoshita, S.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, January 2004, p. 76-86, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.76-86.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Utilization of Escherichia coli Outer-Membrane Endoprotease OmpT Variants as Processing Enzymes for Production of Peptides from Designer Fusion Proteins

Kazuaki Okuno,^1,2* Masayuki Yabuta,¹ Toshihiko Ooi,² and Shinichi Kinoshita²

Institute for Medicinal Research and Development, Daiichi Suntory Pharma Co., Ltd., Akaiwa, Chiyoda-machi, Ohra-gun, Gunma 370-0503,¹ Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-8628, Japan²

Received 18 June 2003/ Accepted 13 October 2003

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp⁹⁷ is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp⁹⁷ were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1'). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Argmotilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.

---

* Corresponding author. Mailing address: Institute for Medicinal Research and Development, Daiichi Suntory Pharma Co., Ltd., 2716-1 Kurakake, Akaiwa, Chiyoda-machi, Ohra-gun, Gunma 370-0503, Japan. Phone: 81-276-86-5784. Fax: 81-276-86-5760. E-mail: Kazuaki_Okuno{at}dsup.co.jp.

---

Applied and Environmental Microbiology, January 2004, p. 76-86, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.76-86.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Varadarajan, N., Gam, J., Olsen, M. J., Georgiou, G., Iverson, B. L. (2005). Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity. Proc. Natl. Acad. Sci. USA 102: 6855-6860 [Abstract] [Full Text]