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-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.
Department of Molecular Biology, ELIAS Entwicklungslabor, Freiburg, Germany.
H
H
Berg
M
M
Walter
L
L
Mauch
J
J
Seissler
W
W
Northemann
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000276:immunology; D001483:Base Sequence; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007328:Insulin; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000276:immunology*; D011498:Protein Precursors / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000302:isolation & purification
D001323:Autoantibodies; D007328:Insulin; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D006639:Histidine; C013737:preproinsulin; D011384:Proinsulin