Purification of a cysteine protease inhibitor from larval hemolymph of the tobacco hornworm (Manduca sexta) and functional expression of the recombinant protein

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• 作者：Takayuki Miyaji ; Yoshiaki Kouzuma ; Jun Yaguchi ; Rika Matsumoto ; Michael R. Kanost ; Karl J. Kramer ; Masami Yonekura
• 关键词：Cysteine protease ; Cysteine protease inhibitor ; Cystatin ; Hemolymph ; Manduca sexta ; Overexpression
• 刊名：Insect Biochemistry and Molecular Biology
• 出版年：2007
• 期刊代码：80_09651748
• 类别：bio
• 出版时间：September 2007
• 卷：37
• 期：9
• 页码：960-968
• 文件大小：656 K

摘要

A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5 kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration on Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a K_i value of 5.5×10⁻⁹ M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrina. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in Escherichia coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (K_i, 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin or themolysin.