Glycoprotein IIIa Preparation for T-cell Stimulation

Abstract

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by antiplatelet autoantibodis. The major target of the anti platelet antibodies is platelet membrane glycoprotein IIb/IIIa. In order to characterize the immunodominant epitopes in the structure of GPIIIa, the extracellular portions of GPIIIa will be expressed and purified. These antigens will be tested for antigenicity in further investigation. The first segment of GPIIIa which was considered for expression as a recombinant glutathion S-transferase (GST) fusion proteins included IIIa22-262 which encompass amino acid residue 22-262 of the 762 amino acids of GPIIIa. A segment of GPIIIa complementary DNA (cDNA) was subcloned into the 39 end of the Schistosoma japonicum GST gene in the bacterial expression plasmid vector, pGEX 6P-1 (Amersham Pharmacia Biotech). In summary, the expression plasmid vector, pGEX 6P-1 containing segment IIIa22-262 was introduced to E.coli. Saturated overnight culture was used and the bacterial cells were grown to log-phase. IPTG was added to the culture to induce overexpression of fusion protein and the cells were grown for an additional 1-3 hours. Bacterial lysate containing recombinant protein was prepared by sonication. The fusion protein was purified from total cell extract using glutathione-agarose beads. Specificity of the GST-fusion proteins was confirmed on Immunoblot probed with rabbit anti-GST polyclonal antibodies. PreScission Protease was used to remove the GST tag. Protein extract and purified products were analyzed by SDS gel electrophoresis. The recombinant GST-fusion protein IIIa22-262 was successfully expressed and purified in large quantities but the yield of the IIIa22-262 peptide after enzyme treatment was low. When a good yield is fully obtained, the purified protein segment will be used for T-cell stimulation in culture.