cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus

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• 作者：Wu ; Fang ; Yan ; Ming ; Li ; Yikun ; Chang ; Shaojie ; Song ; Xiaomin ; Zhou ; Zhaocai ; Gong ; Weimin
• 关键词：Pathogenesis-related ; Ribonuclease ; Mutagenesis ; ANS-binding activity
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2003
• 出版时间：December 19, 2003
• 年：2003
• 卷：312
• 期：3
• 页码：761-766
• 全文大小：397 K

文摘

SPE-16 is a new 16kDa protein that has been purified from the seeds of Pachyrrhizus erosus. It’s N-terminal amino acid sequence shows significant sequence homology to pathogenesis-related class 10 proteins. cDNA encoding 150 amino acids was cloned by RT-PCR and the gene sequence proved SPE-16 to be a new member of PR-10 family. The cDNA was cloned into pET15b plasmid and expressed in Escherichia coli. The bacterially expressed SPE-16 also demonstrated ribonuclease-like activity in vitro. Site-directed mutation of three conserved amino acids E95A, E147A, Y150A, and a P-loop truncated form were constructed and their different effects on ribonuclease activities were observed. SPE-16 is also able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) in the native state. The ANS anion is a much-utilized “hydrophobic probe” for proteins. This binding activity indicated another biological function of SPE-16.