毛果杨半胱氨酸蛋白酶基因PtCP3的基因克隆和原核表达分析

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

为研究毛果杨半胱氨酸蛋白酶基因PtCP3的酶学特征和生理功能,本实验对PtCP3进行了基因克隆和原核表达分析。从毛果杨(Populus trichocarpa Torr.&Gray)中克隆得到半胱氨酸蛋白酶基因PtCP3,将其克隆至pMD-18T载体。进一步构建原核表达载体pET30a-PtCP3,并在大肠杆菌(Escherichia coli)中成功诱导表达重组蛋白,然后通过包涵体洗涤法对目的蛋白进行纯化。测序结果表明,PtCP3基因CDS序列全长1074 bp,含有木瓜蛋白酶的保守催化位点和组织蛋白酶B的保守结构域。序列分析结果显示,半胱氨酸蛋白酶基因PtCP3编码357个氨基酸,预测N-末端含有长度为27个氨基酸残基的信号肽序列,去除信号肽后蛋白酶大小为36.515 kDa,理论等电点为7.44。SDS-PAGE电泳结果显示,可以检测到大小约为42 kDa的目的条带,并可通过包涵体洗涤法在体外得到了高表达量、单一的重组蛋白PtCP3。

The objective was to study the enzymatic characteristics and physiological functions of PtCP3 geneof cysteine protease. PtCP3 gene cloning and prokaryotic expression analysis were conducted. The cysteineprotease gene PtCP3 was cloned from Populus trichocarpa in this research. Pt CP3 was constructed into theexpression vector pMD- 18 T and then constructed the prokaryotic expression vector pET30a- PtCP3. Therecombinant protein was induced and expressed in Escherichia coli successfully. The target protein waspurified by washing method on inclusion body. The sequencing results showed that the full- length CDSsequence of PtCP3 was 1074 bp, and contained the conservative catalytic sites of papain and the conservativestructural domain of cathepsin B. The sequence analysis showed that cysteine protease gene PtCP3 encoded357 amino acids, and predicted N-terminus hydrophobic region containing a signal peptide with 27 amino acidresidues. The protease getting rid of signal peptide was 36.515 k Da, and the isoelectric point was 7.44. TheSDS-PAGE showed that the target band with 42 k Da molecular weight was obtained. The high-express andsingle recombinant protein Pt CP3 was obtained by washing method on inclusion body.