粗糙脉孢菌蛋白表达系统构建研究  

Construction of a Recombinant Protein Expression System in Neurospora crassa

作　　者：高染染 刘倩[2] 孙文良[2] 孙志勇[2] 刘浩[1] 田朝光[2] 

机构地区：[1]天津科技大学生物工程学院教育部工业发酵微生物重点实验室,天津300457 [2]中国科学院天津工业生物技术研究所系统微生物工程重点实验室,天津300308

出　　处：《生物技术通报》2016年第7期160-169,共10页Biotechnology Bulletin

基　　金：国家高技术研究发展计划("863"计划)(SS2012AA023303)

摘　　要：旨在将模式丝状真菌粗糙脉孢菌(Neurospora crassa)构建为蛋白质表达系统。通过真菌杂交技术构建出粗糙脉孢菌六突变缺失菌株LQ-1(Δ3βG∷Δ2cbh∷Δhis3)作为蛋白表达宿主菌株。该宿主菌株可以用纤维二糖作为诱导物诱导纤维素酶启动子表达,同时又消除了葡萄糖苷酶蛋白的背景条带,有利于目标蛋白表达纯化。构建了分别含有粗糙脉孢菌自身强启动子(Pcbh-1、Pcbh-2和Ptef-1)的3个新的高效表达载体,该载体带有融合标签蛋白tev-6×his-gfp,能高效方便的筛选阳性转化子,有利于后续目标蛋白纯化。以纤维素酶GH3-4和CBH-1为例,通过重组表达菌株纤维二糖诱导发酵液进行酶活测定、SDS-PAGE电泳分析和Western blotting检测显示,重组蛋白GH3-4-GFP和CBH-1-GFP成功进行了表达和分泌,分泌水平分别为2.77和2.83 mg/L。Pcbh-1启动子重组蛋白表达水平最高,说明在纤维二糖诱导体系中启动子Pcbh-1的启动效率最高,初步建立了粗糙脉孢菌纤维二糖诱导的蛋白质表达体系。To demonstrate the potential of the model filamentous fungus Neurospora crassa as a recombinant protein expression system,we used fungal hybrid technology to develop a sextuple gene disruptant LQ-1（Δ3βG∷Δ2cbh∷Δhis3）of it as the host strain of expressingthe protein.The cellobiose was used to induce the expression of cellulase promoter in host strain,which removed the background band ofglucosaccharase and was conducive to the expression and purification of target protein.We constructed 3 new efficiently-expressed vectorsrespectively consisting of its own strong inducible promoters（Pcbh-1,Pcbh-2 and Ptef-1）from N.crassa.Those vectors had fusion tag proteintev-6×his-gfp and efficiently and conveniently screened positive transformants,which was favourable for the purification of later target protein.Two endogenous cellulase（GH3-4 and CBH-1）were chosen as test proteins for recombinant expression.Then enzymatic activity measuredby cellobiose-induced fermentation broth of recombinant strain,SDS-PAGE analysis,and Western blotting indicated that the recombinantproteins GH3-4-GFP and CBH-1-GFP were successfully expressed with secreted levels reaching as high as approximate 2.77 and 2.83 mg/L.The expression of recombinant protein with Pcbh-1 promoter was the highest,suggesting that promoting efficiency of Pcbh-1 in the cellobioseinduced system was the highest,and a protein expression system in cellobiose-induced N.crassa was preliminarily constructed.

关 键 词：丝状真菌 粗糙脉孢菌 蛋白表达系统 纤维二糖 强启动子 

分 类 号：Q78[生物学—分子生物学]