Testing an alternative expression system for protozoan proteins

PETRIGH, R; FARBER, M

INTA - Castelar, Buenos Aires

Jornada; BABESIA WORLD SUMMIT; 2005

CICVyA - INTA

The expression of genes in environments that allow the production of functional proteins that can be purified for further applications constitute a main foal in order to improve the current expresión tools for obtaining recombinant proteins. To pusue that achievement we are setting up a novel expression system using Chritidia fasciculata. The evolutionary proximity of C. fasciculate to important human and animal pathogens, such as Trypanosoma cruzi, Leishmania, Toxoplasma gondii, Plasmodium falciparum and Babesia sp., makes them an ideal vehicle for the expression of genes from these other organisms. The system should represent and excellent alternative for expressing protozoan proteins. Crithidia fasciculata is a non-pathogenic parasite of insects which can be cultivated in high yields using inexpensive undefined serum-free media and does not require specific bio-safety precautions. The most important point is that the basic cellular machinery of Crithidia is an approach for the study of protein folding an glycosilation in Babesia sp. Expression vectors containing a promoter rRNA and a 5’ and 3’ UTR sequence from C. fasciculate were used. Constructs include poly-histidine tags to facilitate purification, green fluorescence protein (GFP) to enable localization of fusion proteins or a secretion peptide signal to recover the protein in the medium. C. fasciculate was grown in Brain Heart Infusion (BHI)with haemin. To improve transfection efficiency, several conditions of electroporation were evaluated using the construct with GFP. Transfected parasites were selected with Hygromycin B and resistant lines were obtained after 3 weeks. Up to now we cloned msa2c gene from Babesia bovis in the expression vectors. Stable lines were obtained and expression of MSA2c was achieved.