Bossé M, Handl C E, Lortié L A, Harel J, Dubreuil J D
Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Montréal University, St-Hyacinthe, Québec, Canada.
J Gen Microbiol. 1993 Mar;139(3):631-8. doi: 10.1099/00221287-139-3-631.
The heat-stable enterotoxin b gene (estB) of Escherichia coli was fused to the gene for maltose-binding protein (malE). The estB gene was cloned into the pMAL-p vector using PCR. The construct consists of the signal sequence of maltose-binding protein, which directs the export of the fusion protein to the periplasm, and the maltose-binding protein fused to the STb polypeptide. A sequence encoding a factor Xa cleavage site is present between malE and estB. The fused genes are controlled by Ptac, a strong inducible promoter. Following IPTG induction, the recombinant strain expressed a 47 kDa protein, which was easily purified from osmotic shock fluid by using preparative electrophoresis and electroelution. Cleavage of the fusion protein with factor Xa generated the maltose-binding protein (42 kDa) and a polypeptide of approximately 5 kDa, corresponding to the molecular mass of mature STb. A monospecific polyclonal rabbit antiserum raised against purified STb reacted in immunoblot with the fusion protein and the cleaved-off peptide. A positive response was observed when testing the osmotic shock fluid containing the fusion protein in a rat intestinal loop assay. On average, 3-4 mg of MBP-STb protein was recovered per litre of induced recombinant strain.
大肠杆菌的热稳定肠毒素b基因（estB）与麦芽糖结合蛋白（malE）基因融合。使用聚合酶链反应（PCR）将estB基因克隆到pMAL-p载体中。构建体由麦芽糖结合蛋白的信号序列组成，该信号序列指导融合蛋白输出到周质，以及与STb多肽融合的麦芽糖结合蛋白。在malE和estB之间存在编码因子Xa切割位点的序列。融合基因由强诱导型启动子Ptac控制。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，重组菌株表达了一种47 kDa的蛋白，该蛋白可通过制备电泳和电洗脱从渗透休克液中轻松纯化。用因子Xa切割融合蛋白产生了麦芽糖结合蛋白（42 kDa）和一条约5 kDa的多肽，其分子量与成熟STb相对应。用针对纯化的STb产生的单特异性多克隆兔抗血清在免疫印迹中与融合蛋白和切割下来的肽发生反应。在大鼠肠袢试验中检测含有融合蛋白的渗透休克液时观察到阳性反应。每升诱导的重组菌株平均回收3 - 4 mg的MBP-STb蛋白。
