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J. Microbiol. Biotechnol. 2004; 14(2): 417-421

Published online April 28, 2004

Copyright © The Korean Society for Microbiology and Biotechnology.

Cloning, Sequencing, and Expression of cDNA Encoding Bovine Prion Protein

Kang Sang Gyun , Sung Keun Kang 1, Deog Yong Lee 1, Yong Ho Park 2, Woo Suk Hwang 2 and Han Sang Yoo 2*

Department of Infectious Disease College of Veterinary Medicine and School of Agricultural Biotechnology Seoul National University San 56-1 Shinlim-dong Kwanak-gu Seoul 151-742 Korea, 1Theriogenology and Biotechnology College of Veterinary Medicine and School of Agricultural Biotechnology Seoul National University San 56-1 Shinlim-dong Kwanak-gu Seoul 151-742 Korea, 2Theriogenology and Microbiology College of Veterinary Medicine and School of Agricultural Biotechnology Seoul National University San 56-1 Shinlim-dong Kwanak-gu Seoul 151-742 Korea

Abstract

A normal prion protein (PrPc) is converted to a protease resistant isoform (PrPsc) by an apparent self-propagating activity in bovine spongiform encephalopathies (BSE), which is a neurodegenerative disease. The cDNA encoding bovine PrP open reading frame (ORP) in Korean cattle was cloned by polymerase chain reaction (PCR). The cloned cDNA had a length of 795 base pairs which coded for a protein of 264 amino acid residues with a calculated molecular mass of 28.6 kDa. Identities of 90, 90, 79 and 78% on nucleotide and 94, 94, 84, and 84% on amino acid sequence were shown to PrP genes from sheep, goat, human, and mouse, respectively. The cloned DNA was ligated into the pQE30 expression vector and transformed into E. coli M15. The PrP was expressed by induction with isopropyl-$\beta$-D-thiogalactoside (IPTG) and purified on the Ni-NTA affinity column. High specific activities of the recombinant PrP were observed in the fraction of pH 5.8 eluate and showed a molecular mass of-29 kDa on SDS-PAGE and Western blot analysis.

Keywords: Prionprotein, cDNA, BSE, Koreancattle