Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety ‘‘Golden Valley’’ Mi-Hyun Kim a , Seong-Cheol Park a,e , Jin-Young Kim a , Sun Young Lee a , Hak-Tae Lim d , Hyeonsook Cheong c , Kyung-Soo Hahm a,b, * , Yoonkyung Park a,c, * a Research Center for Proteineous Materials (RCPM), Chosun University, Kwangju 501-759, Republic of Korea b Department of Medicine, Chosun University, Kwangju 501-759, Republic of Korea c Department of Biotechnology, Chosun University, Kwangju 501-759, Republic of Korea d Division of Biotechnology, Kangwon National University, 192-1, Hyoja2-Dong, Chunchon, Kangwon-Do 200-701, Republic of Korea e Division of Applied Life Science, Gyeongsang National University, Chinju 660-701, Republic of Korea Received 18 May 2006 Available online 9 June 2006 Abstract Potide-G, a small (5578.9 Da) antimicrobial peptide, was isolated from potato tubers (Solanum tuberosum L. cv. Golden Valley) through extraction of the water-soluble fraction, dialysis, ultrafiltration and DEAE-cellulose and C 18 reversed-phase high performance liquid chromatography. This antimicrobial peptide was heat-stable and almost completely suppressed the proteolytic activity of trypsin, chymotrypsin and papain, with no hemolytic activity. In addition, potide-G potently inhibited growth of a variety of bacterial (Staph- ylococcus aureus, Listeria monocytogenes, Escherichia coli, and Clavibacter michiganense subsp. michiganinse) and fungal (Candida albi- cans and Rhizoctonia solani) strains. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the N-terminal sequence (residues from 1 to 11) of the protein is identical to that of potato proteinase inhibitor, a member of the Kunitz superfamily. And like other members of this class of protease inhibitor, potide-G may have number of beneficial and ther- apeutic uses. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Antimicrobial peptide; Proteinase inhibitor; Inhibition activity; Perianal dermatitis Plants express a variety of proteins that serve in the defense against pathogens and invading organisms, includ- ing ribosome-inactivating proteins [1], lectins [2], protease inhibitors [3] and antifungal proteins [4–6]. Among these, protease inhibitors are believed to play an important role in the defense against attack by both microorganisms and insects, to serve as storage proteins and, perhaps, to be involved in the regulation of endogenous proteases during seed dormancy [7]. Among the different types of protease inhibitors that have been identified in plants [8], particular- ly high levels of inhibitors of proteinases are found in many Solanaceae family members [9] and account for as much as 20–50% of the water-soluble proteins in potato tubers [10], which is noteworthy, as increasing levels of protease inhib- itor correlates with increases in resistance to pathogens [11]. Indeed, these peptides have recently attracted atten- tion because of their ability to potently inhibit carcinogen- esis [12] as well as the growth of pathogenic bacteria and fungus in a number of in vitro systems [13]. In this report, we describe the isolation and character- ization of a small antimicrobial peptide, potide-G, from Golden Valley potato tubers. This peptide exhibits potent inhibitory activity against various human or plant patho- genic microbial strains and shows sequence homology with a protease inhibitor that is produced by potato tubers upon fungal attack [13]. Interestingly, the N-termi- nal sequence of potide-G is identical to the corresponding sequence of proteinase inhibitor, a member of the Kunitz superfamily. 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.186 * Corresponding authors. Fax: +82 62 2278345. E-mail address: kshahm@chosun.ac.kr (K.-S. Hahm). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 346 (2006) 681–686 BBRC