毛果杨半胱氨酸蛋白酶基因PtCP2和PtCP4原核表达及蛋白纯化

Prokaryotic expression and protein purification of cysteine protease genes PtCP2 and PtCP4 from Populus trichocarpa Torr. & Gray

【目的】对毛果杨半胱氨酸蛋白酶基因Pt CP_2和Pt CP4进行原核表达及重组蛋白纯化,为进一步研究该基因的酶学特征和生理功能打下基础。【方法】从毛果杨中克隆两个半胱氨酸蛋白酶基因Pt CP_2和Pt CP4,构建其原核表达载体p ET-30a-Pt CP_2及p ET-30a-Pt CP4,并在转入大肠杆菌后在体外诱导表达重组蛋白,采用包涵体洗涤法对目的蛋白进行纯化。【结果】Pt CP_2基因CDS序列全长1107 bp,编码368个氨基酸;Pt CP4基因CDS序列全长1104 bp,编码367个氨基酸。Pt CP_2和Pt CP4基因编码的蛋白均属于RD19A-like类型木瓜蛋白酶。Pt CP_2和Pt CP4基因在毛果杨根、茎、叶中均有表达,其中Pt CP_2在叶中表达量最高,Pt CP4在根中表达量最高。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Pt CP_2和Pt CP4,切除信号肽后蛋白酶大小分别为38.151和38.069 k D。【结论】原核表达获得的纯化重组蛋白Pt CP_2和Pt CP4可用于进一步研究毛果杨半胱氨酸蛋白酶的酶学特征和生理功能。

[Objective]The present study was conducted to express cysteine protease genes PtCP2 and PtCP4 of P. tr/- chocarpa Torr. ＆ Gray in the prokaryotic cell, and purify its recombinant proteins, in order to lay the foundation for zymo- logical characteristics and physiological functions of PtCP2 and PtCP4 in P. trichocarpa. [Method]Two cysteine protease genes PtCP2 and PtCP4 were cloned from P. tdchocarpa, which were linked to vector pET-3Oa（t）, so as to construct prokaryotic expression vectors pET-30a-PtCP2 and pET-3Oa-PtCP4. Then prokaryotic expression vectors were transferred into Esehetiehia eoli. And the recombinant proteins were obtained by induction, and then purified by washing inclusion body.[Result]The results showed that, the PtCP2 gene CDS was 1107 bp in length, encoding 368 amino acids. And PtCP4 gene CDS was 1104 bp in length, encoding 367 amino acids. Both of them belonged to RD19A-like type of papain. Real- time PCR results showed, the PtCP2 and PtCP4 were expressed in stem, leaves, and roots of 1-year-old P. tdchocarpa. The PtCP2 gene was expressed highly in the leaves, however PtCP4 gene was expressed highly in the roots. The high-purity recombinant proteins PtCP2 and PtCP4 could be obtained by washing inclusion body/n vitro, and which were 38.151 and 38.069 kD in molecular weight after removal of signal peptide. [Conclusion]The recombinant proteins PtCP2 and PtCP4 obtained in vitro can be applied to study enzymatic characteristics and physiological functions of cysteine protease in P. trichocarpa.