Molecular cloning, comparative sequence analysis and prokaryotic expression of GRA5 protein of Toxoplasma gondii*

Abstract

Among the several target molecules for sensitive detection of Toxoplasma gondii, dense granule antigens are considered important as these help in growth and multiplication of the organism in the host. The communication deals with the cloning and sequence analysis of 363 bp entire open reading frame (ORF) of GRA5, a dense granule protein, from 2 Indian isolates of T. gondii (Izatnagar and Chennai) as well as the standard RH strain. The sequence comparison analysis revealed 100% homology between the Chennai and Izatnagar isolates, 99.2% homology of RH strain with both the Chennai and Izatnagar isolates and 100% sequence homology of RH strain of T. gondii with the published sequence. The GRA5 protein (mature) was subsequently expressed in prokaryotic expression system. It had molecular size of ~29 kDa and the level of expression was measured as 12% of the total protein. The concentration of the mature recombinant GRA5 protein was 92 μg/ml. Western blot with Ni-NTA anti-histidine HRPase conjugate and known positive serum confirmed the presence and purity of protein by immunoreactivity at the unique ~29 kDa region.