Proteomic of the nucleoside diphosphate kinase from T. cruzi: preliminary estructural studies, expression and purification

GOMEZ BARROSO JA; MIRANDA MR; PEREIRA CA; AGUILAR CF

Misiones

Congreso; XL Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2004

The enzyme nucleoside diphosphate kinase (NDPK) is a majorm component of the pathway for the synthesis of nucleoside triphosphates other than ATP. The mechanism of reaction involves the formation of a phospho-histidine intermediate. All known NDPK are oligomers made of small polypeptides of about 150 residues (17kDa) with a high degree of sequence similarity. T.cruzi NDPK (TcNDPK) has been characterized and could be involved in flagellar movement and therefore in the pathology of the parasite. T. cruzi NDPK (TcNDPK) has been cloned in E. coli DH5á strain. The gene has been cloned into a pRSET A plasmid and expressed in E. coli BL21-DE3 strain as a fusion protein containing TcNDPK preceded by an hexa-His-tag. The fusion protein has been purified with a HiTrap chelating column loaded with 0,1M NiSO4 in different buffer conditions and its purification level visualized in SDS-PAGE. The conformational state of the purified TcNDPK has been observed by different methods such as native PAGE. We have obtained large quantities of TcNDPK to be used for crystallization and interaction assays. The objective of this work is the resolution of the three-dimentional structure of TcNDPK by X-ray crystallography as a first step for rational drug design based on the structure.