海栖热袍菌极耐高温木聚糖酶基因xynB64在大肠杆菌中的融合表达

Fusion expression of an extreme-thermostable xylanase B64 gene from Thermotoga maritima MSB8 in Escherichia coli

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。

Abstract:

Xylanase B from thermophile bacteria Thermotoga maritima MSB8 has extreme-thermostability, which has potential widely application for feed, papermanufacture, energy, food and medicine industries. Recombinant pET28a(+)-xynB64 was induced and expressed in E. coli BL21(DE3), and the activity 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.