Chinery R, Poulsom R, Elia G, Hanby A M, Wright N A
Imperial Cancer Research Fund/Royal College of Surgeons Histopathology Unit, London, England.
Eur J Biochem. 1993 Mar 1;212(2):557-63. doi: 10.1111/j.1432-1033.1993.tb17693.x.
The cysteine-rich trefoil motif of rat intestinal trefoil factor (rITF) was cloned and expressed in Escherichia coli. A 270-bp cDNA fragment including the signal sequence and the trefoil motif was cloned into the expression vector pAX5+ to direct the expression of a beta-galactosidase collagen-hinged fusion protein in E. coli. Cultures harbouring the recombinant plasmid produced a soluble novel protein with a molecular mass of 134.5 kDa, as predicted for the trefoil-motif-containing fusion protein. Purification of the rITF moiety was achieved by p-aminophenyl-thio-beta-D-galactoside(APTG)-affinity chromatography, collagenase digestion of the hybrid molecule, and removal of the beta-galactosidase-hinge molecule by a further APTG-affinity step. It was demonstrated that intrachain disulphide-bond formation in rITF occurred during the procedure, so no refolding steps were required. Analysis by immunoblotting revealed that the fusion protein and the cleaved trefoil-motif-containing protein were recognised by an antibody raised against the chemically synthesised peptide. The trefoil motif present in the fusion protein was used to localise putative trefoil-binding sites in sections of frozen rat tissue. Binding was demonstrated using the beta-galactosidase portion of the fusion protein as a reporter moiety, either directly with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside, or indirectly using a monoclonal antibody to beta-galactosidase and indirect immunohistochemistry. Binding sites were localised to the foveolar and surface epithelium of rat stomach, the collecting ducts of the kidney and within colonic crypts. The presence of a trefoil motif was necessary for binding. The use of beta-galactosidase fusion proteins for histochemical localisation of peptide-binding sites should prove more generally useful.
大鼠肠三叶因子（rITF）富含半胱氨酸的三叶模体被克隆并在大肠杆菌中表达。一个包含信号序列和三叶模体的270 bp cDNA片段被克隆到表达载体pAX5 +中，以指导β-半乳糖苷酶胶原铰链融合蛋白在大肠杆菌中的表达。携带重组质粒的培养物产生了一种可溶性新蛋白，其分子量为134.5 kDa，这与含三叶模体的融合蛋白的预测分子量一致。通过对氨基苯硫基-β-D-半乳糖苷（APTG）亲和层析、胶原酶消化杂合分子以及通过进一步的APTG亲和步骤去除β-半乳糖苷酶铰链分子，实现了rITF部分的纯化。结果表明，rITF中的链内二硫键形成发生在该过程中，因此不需要复性步骤。免疫印迹分析表明，融合蛋白和切割后的含三叶模体蛋白可被针对化学合成肽产生的抗体识别。融合蛋白中存在的三叶模体被用于在冷冻大鼠组织切片中定位假定的三叶结合位点。使用融合蛋白的β-半乳糖苷酶部分作为报告基团，直接用5-溴-4-氯-3-吲哚基-β-D-半乳糖苷，或间接使用抗β-半乳糖苷酶单克隆抗体和间接免疫组织化学方法，证明了结合的存在。结合位点定位于大鼠胃的小凹和表面上皮、肾集合管以及结肠隐窝内。三叶模体的存在是结合所必需的。使用β-半乳糖苷酶融合蛋白进行肽结合位点的组织化学定位应具有更广泛的用途。
