草菇漆酶基因vv-lac1和vv-lac6的克隆及异源表达

Cloning and heterologous expression of laccase genes vvlac1 and vv-lac6 from Volvaria volvacea

摘要:

摘要:【目的】克隆草菇漆酶基因vv-lac1和vv-lac6的全长cDNA，证实其编码的蛋白具有漆酶活性，最终建立草菇漆酶基因的异源表达及纯化体系。【方法】利用RACE技术克隆全长cDNA序列，并利用生物信息学技术进行序列分析，在此基础上，去除全长cDNA的编码信号肽的序列并在3'端添加His-tag碱基序列，把修饰后的cDNA片段克隆到表达载体pPIC9K上，并转化到毕赤酵母GS115中进行表达，对重组蛋白利用Ni柱进行分离纯化并以ABTS底物法检测重组蛋白的漆酶活性。【结果】vv-lac1和vv-lac6的全长cDNA的长度分 别为1599 bp和1554 bp，且分别含有19和15个外显子;其编码的蛋白的理论分子量分别是57.3 kDa和56.3 kDa，理论等电点分别为4.73和5.62，且都属于分泌型的胞外蛋白;表达出的重组蛋白RBvvlac1和RBvvlac6，其分子量大小约为70 kDa，说明存在翻译后修饰;含有150 mmol/L咪唑的缓冲液对RBvvlac1和RBvvlac6发酵液进行洗脱所得到的蛋白溶液具有最高漆酶活性(333.17 U/L和227.63 U/L)。【结论】草菇漆酶基因vv-lac1和vv-lac6能够编码有活性的漆酶蛋白，本研究建立的异源表达及纯化体系适用于草菇或 其它漆酶基因的异源表达与纯化。

关键词:关键词:草菇，漆酶基因，异源表达，漆酶活性

Abstract:

Abstract:［Objective］To clone the full-length cDNAs of two laccase genes，vv-lac1and vv-lac6，from Volvaria volvacea，verify their encoded proteins with laccase activity and develop a heterologous expression and protein purification system for V．volvacea laccase genes.［Methods］The full-length cDNAs were cloned with rapid amplification of cDNA ends (RACE) technology and carried out in silico analysis．After modified by removing the sequence encoding signal peptide and adding the sequence encoding His-tag at 3' ends，the cDNAs were cloned into pPIC9K vector．The resulting constructs were transformed into Pichia pastoris GS115 for heterologous expression．The recombinant proteins were purified with Ni columns and the laccase activity were detected with ABTS assay.［Results］The full-length cDNAs of vv-lac1 and vv-lac6 are 1，599 bp and 1，554 bp，and contain19 and 15 exons，respectively．The predicted molecular weights of the proteins encoded by vv-lac1 and vv-lac6 are 57.3 kDa and 56.3 kDa，respectively．The predicted isoelectric points are 4.73 and 5.62，respectively．Both proteins are extracellular．The recombinant proteins RBvvlac1and ＲBvvlac6 are 70kDa，which may be modified by posttranslational modification．The solutions of the two recombinant proteins eluted by 150 mmol/L imidazole eluent have the highest laccase activity levels (333.17 U/L and 227.63 U/L).［Conclusion］The proteins encoded by the laccase genes vv-lac1and vv-lac6 from V．volvacea have laccase activity，the heterologous expression and protein purification system developed in this study is suitable for future studies of other laccase genes from V．volvacea or other fungi．

Key words:Keywords: Volvaria volvacea，laccase gene，heterologous expression，laccase activity