海栖热袍菌极耐高温木聚糖酶基因xynB_(64)在大肠杆菌中的融合表达  

Fusion expression of an extreme-thermostable xylanase B_(64) gene from Thermotoga maritima MSB8 in Escherichia coli

作　　者：孙涛[1] 申宁[1] 白羽[1] 李文豪[1] 韦萍[1] 

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京210009

出　　处：《微生物学通报》2011年第7期1090-1097,共8页Microbiology China

基　　金：国家科技支撑计划重点项目(No.2008BAI63B07)

摘　　要：来源于极端嗜热菌海栖热袍菌(Thermotoga maritima MSB8)的木聚糖酶B具有极高的热稳定性,在饲料、造纸、能源和食品医药行业具有巨大应用潜力。携带酶基因xynB64的pET28a(+)重组载体在宿主大肠杆菌BL21(DE3)中诱导表达,重组酶活力较低。更换宿主为携带稀有tRNA基因的大肠杆菌:BL21-CodonPlus(DE3)-RIPL和Rosetta(DE3)后,酶活力分别提高了197%和277%,但是后者中的表达会形成部分包涵体。宿主菌为大肠杆菌Rosetta(DE3),更换载体为4种融合表达载体pET32a(+)、pET42a(+)、pET43.1a(+)和pMAL-c2X进行表达,重组酶分别融合了Trx、GST、Nus和MBP标签。其中Rosetta(DE3)/pMAL-c2X-xynB64表达酶活力最高,相当于Rosetta(DE3)/pET28a-xynB64表达酶的88%,而且目的酶表达量占全细胞蛋白的40%,几乎不形成包涵体。Xylanase B from thermophile bacteria Thermotoga maritima MSB8 has extreme-thermostability,which has potential widely application for feed,papermanufacture,energy,food and medicine indus-tries.Recombinant pET28a（＋）-xynB64 was induced and expressed in E.coli BL21（DE3）,and the activ-ity of recombinant XynB64 was very low.E.coli BL21-CodonPlus（DE3）-RIPL and Rosetta（DE3） both harbouring rare tRNAs were used to replace E.coli BL21（DE3） and the activity of recombinant XynB64 increased by 197% and 277%,respectively.However,some inclusion body was formed in E.coli Rosetta（DE3）.Next,pET32a（＋）,pET42a（＋）,pET43.1a（＋） and pMAL-c2X,which has the Trx,GST,Nus and MBP fusion tag respectively were used to replace pET28a（＋） with E.coli Rosetta（DE3） as host.The activity of recombinant XynB64 produced by Rosetta（DE3）/pMAL-c2X-xynB64 was highest,which was equivalent to 88% of counterparts of Rosetta（DE3）/pET28a-xynB64.Meanwhile about 40 percent whole cell proteins of former were recombinant XynB64 with little inclusion body.

关 键 词：耐高温木聚糖酶 稀有密码子 融合标签 翻译起始区 

分 类 号：Q78[生物学—分子生物学]