State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Plasmid. 2013 Sep;70(2):272-6. doi: 10.1016/j.plasmid.2013.05.003. Epub 2013 May 20.
To express homologous or heterologous proteins in fungi, a protein expression system using the promoter of cellobiohydrolase II gene (cbhII) was constructed by generating an expression vector called pWEIIF00. The obtained vector possesses the left and right borders, a hygromycin phosphotransferase B selective marker and a strong promoter and terminator of cbhII from Trichoderma reesei. It can easily undergo random recombination. The applicability of the vector was tested by red fluorescent protein gene (DsRed2) expression detection in T. reesei Rut C30. Using this system, a recombinant Cel5A variant, N342R (Qin et al., 2008), was then selected to express in Rut-C30. Compared to that of the parent strain, integration of the N342R gene resulted in 31.09% increased carboxymethyl-cellulose-degrading (CMCase) activity at pH 5.0 and 56.06% increased activity at pH 6.0. The increased CMCase activity of the recombinant strains would be beneficial for its application uses in multiple industries. The vector constructed in this study can used in fungi to produce industrial proteins.
为了在真菌中表达同源或异源蛋白，通过生成一个名为 pWEIIF00 的表达载体，构建了使用纤维二糖水解酶 II 基因（cbhII）启动子的蛋白表达系统。该获得的载体具有左右边界、潮霉素磷酸转移酶 B 选择性标记以及来自里氏木霉的 cbhII 的强启动子和终止子。它可以很容易地进行随机重组。该载体的适用性通过在里氏木霉 Rut C30 中检测红色荧光蛋白基因（DsRed2）的表达来测试。使用该系统，选择表达重组 Cel5A 变体 N342R（Qin 等人，2008 年）。与亲本菌株相比，N342R 基因的整合导致在 pH 5.0 时羧甲基纤维素降解（CMCase）活性增加 31.09%，在 pH 6.0 时活性增加 56.06%。重组菌株的增加的 CMCase 活性将有利于其在多个行业中的应用。本研究中构建的载体可用于真菌生产工业蛋白。
