Berg H, Walter M, Mauch L, Seissler J, Northemann W
Department of Molecular Biology, ELIAS Entwicklungslabor, Freiburg, Germany.
J Immunol Methods. 1993 Sep 15;164(2):221-31. doi: 10.1016/0022-1759(93)90315-x.
A novel prokaryotic expression vector pGEX-6T was designed for high-level expression of recombinant fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant human preproinsulin at its C-terminus. Efficiency of expression was investigated in the Escherichia coli strain CAG456. The synthesized protein was sequestered in an insoluble form in inclusion bodies and was purified to homogeneity by one-step affinity chromatography based on the specific complex formation of the histidine-hexapeptide and a chelating matrix which was charged with Ni2+ ions. The antigenic nature of the purified recombinant preproinsulin fusion protein was evaluated by ELISA screening for insulin autoantibodies in selected sera from patients with recent-onset type 1 (insulin-dependent) diabetes mellitus classified by the existence of additional autoantibodies reactive against glutamic acid decarboxylase. 14% of the tested sera (n = 43) contained insulin autoantibodies which strongly recognized the recombinant human preproinsulin. Comparable measurements with both recombinant human preproinsulin and mature insulin suggested that the observed autoantigenicity of preproinsulin was mediated by the C-peptide or/and signal peptide.
设计了一种新型原核表达载体pGEX-6T，用于高效表达重组融合蛋白，该融合蛋白的N端为组氨酸六肽和谷胱甘肽-S-转移酶，C端为重组人胰岛素原前体。在大肠杆菌菌株CAG456中研究了表达效率。合成的蛋白以不溶性形式隔离在包涵体中，并基于组氨酸六肽与负载Ni2+离子的螯合基质的特异性复合物形成，通过一步亲和层析纯化至同质。通过ELISA筛选近期发病的1型（胰岛素依赖型）糖尿病患者的选定血清中的胰岛素自身抗体，评估纯化的重组胰岛素原前体融合蛋白的抗原性质，这些患者根据是否存在针对谷氨酸脱羧酶的其他自身抗体进行分类。14%的测试血清（n = 43）含有强烈识别重组人胰岛素原前体的胰岛素自身抗体。对重组人胰岛素原前体和成熟胰岛素的类似测量表明，观察到的胰岛素原前体的自身抗原性是由C肽或/和信号肽介导的。
