Heterologous Expression, Purification, and Properties of a Potato Protein Inhibitor of Serine Proteinases

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A. S. Speranskaya¹, A. A. Krinitsina², T. A. Revina¹, N. G. Gerasimova¹, Ya. S. Keruchen'ko¹, A. B. Shevelev¹, and T. A. Valueva^1*

¹Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071 Moscow, Russia; fax: (495) 954-2732; E-mail: valueva@inbi.ras.ru

²Faculty of Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

^* To whom correspondence should be addressed.

Received June 13, 2006; Revision received June 30, 2006

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The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.

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KEY WORDS: Kunitz-type proteinase inhibitors, serine proteinases, potato, heterologous expression

DOI: 10.1134/S0006297906110022

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