RECOMBINANT HUMAN PREPROINSULIN - EXPRESSION, PURIFICATION AND REACTION WITH INSULIN AUTOANTIBODIES IN SERA FROM PATIENTS WITH INSULIN-DEPENDENT DIABETES-MELLITUS

A novel prokaryotic expression vector pGEX-6T was designed for high-le vel expression of recombinant fusion protein with a histidine-hexapept ide and glutathione-S-transferase at its N-terminus and the recombinan t human preproinsulin at its C-terminus. Efficiency of expression was investigated in the Escherichia coli strain CAG456. The synthesized pr otein 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 chel ating matrix which was charged with Ni2+ ions. The antigenic nature of the purified recombinant preproinsulin fusion protein was evaluated b y ELISA screening for insulin autoantibodies in selected sera from pat ients with recent-onset type 1 (insulin-dependent) diabetes mellitus c lassified by the existence of additional autoantibodies reactive again st glutamic acid decarboxylase. 14% of the tested sera (n = 43) contai ned insulin autoantibodies which strongly recognized the recombinant h uman preproinsulin. Comparable measurements with both recombinant huma n preproinsulin and mature insulin suggested that the observed autoant igenicity of preproinsulin was mediated by the C-peptide or/and signal peptide.