PMCA subcloning and expression in a insect stable cell line

CANDELARIA DE LA FUENTE; HUGO ADAMO; ROSSI, J.P.F.C.

Mar del Plata, Argentina

Congreso; XLIII Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The mammalian plasma membrane calcium pump (PMCA) is a P- ATPase which extrudes Ca2+ from the cell using energy obtained from the hydrolysis of ATP. PMCA is encoded by 4 different genes, and these genes can be alternatively spliced, creating different splice variants. Our aim is to express PMCA isoform h4b in an insect stable cell line. In this way, high amount of biologically active PMCA will be obtained, which will be used to study variations in its expression in comparison with that transiently expressed. To carry out our study, PMCA h4b was subcloned in a vector containing an early and hard promoter (OpiE2) from Baculovirus, specific for insect cells. The plasmid pSG5 containing the PMCA h4b sequence was digested to release the PMCA DNA sequence, which was then subcloned in the plasmid pIB/V5 His Topo, previously digested. Ultracompetent cells were transformed with the plasmid pIB/V5 His Topo containing the PMCA isoform. The plasmids were purified from the colonies and digested with the enzymes used to clone the isoform. The products of enzyme digestion showed a molecular weight equal to the corresponding isoform.  We are carrying out transfections of Sf9 insect cells with this plasmid using liposome. PMCA expression will be confirmed by Western-blot. The enzyme will be purified from these cells and then characterized by kinetic and structural studies