EXPRESSION AND CHARACTERIZATION OF LENS MEMBRANE INTRINSIC PROTEIN, MIP, IN A BACULOVIRUS EXPRESSION SYSTEM

Purpose. Major intrinsic protein (MIP) is the transmembrane protein in the lens involved in homeostasis of the fiber cell. Although MIP has some intrinsic water and glycerol permeability and call form a gap jun ction like channels in the reconstituted systems, its role in the lens remained enigmatic. This study is aimed at developing a heterologous expression system for understanding the role of MIP. Methods. The codi ng sequence of bovine MIP was subcloned into pBlueBacIII Baculovirus t ransfer vector to facilitate transfer to AcMNPV genome, and the recomb inant Baculovirus was used to transfect Sf9 cells, Expression of MIP w as confirmed by immunoblotting and purified by HPLC. Reconstituted lip osomes were used to study the function of the recombinant protein. Res ults. SDS-PAGE and immunoblots confirmed that the expressed protein wa s targeted to the cell membrane. MIP accounted for about 10% of total membrane proteins of the transfected Sf9 cell membranes, Molecular siz e of the recombinant protein is estimated to be about 30-32 kDa on SDS -PAGE, in contrast to 26 kDa of native MIP. The reconstitution. studie s showed that the channels formed by recombinant MIP are functionally similar to those of native MIP isolated from calf lens membranes, Conc lusions. This study describes functional expression of MIP in a Baculo virus expression system, This opens the way to studying the rule of MI P and to understanding the effect af mutations of critical residues in the function of MIP.