Cloning of Cysteine Protease Gene PtCP3 and Its Prokaryotic Expression in Populus trichocarpa Torr. & Gray

doi:10.11924/j.issn.1000-6850.casb16010111

Abstract

Abstract: The objective was to study the enzymatic characteristics and physiological functions of PtCP3 gene of cysteine protease. PtCP3 gene cloning and prokaryotic expression analysis were conducted. The cysteine protease gene PtCP3 was cloned from Populus trichocarpa in this research. PtCP3 was constructed into the expression vector pMD-18T and then constructed the prokaryotic expression vector pET30a-PtCP3. The recombinant protein was induced and expressed in Escherichia coli successfully. The target protein was purified by washing method on inclusion body. The sequencing results showed that the full-length CDS sequence of PtCP3 was 1074 bp, and contained the conservative catalytic sites of papain and the conservative structural domain of cathepsin B. The sequence analysis showed that cysteine protease gene PtCP3 encoded 357 amino acids, and predicted N-terminus hydrophobic region containing a signal peptide with 27 amino acid residues. The protease getting rid of signal peptide was 36.515 kDa, and the isoelectric point was 7.44. The SDS-PAGE showed that the target band with 42 kDa molecular weight was obtained. The high-express and single recombinant protein PtCP3 was obtained by washing method on inclusion body.

CLC Number:

Yao Xiaoyun,Zhang Qiang,Han Jingyi,Wang Ying,Cao Shan,Jiang Luyao,Li Lihong,Li Hui and Lu Hai. Cloning of Cysteine Protease Gene PtCP3 and Its Prokaryotic Expression in Populus trichocarpa Torr. & Gray[J]. Chinese Agricultural Science Bulletin, 2016, 32(14): 50-55.

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