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Expression and characterization of lens membrane intrinsic protein, MIP, in a baculovirus expression system
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA. smruthin@mail.mcg.edu
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 can form a gap junction 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 coding sequence of bovine MIP was subcloned into pBlueBacIII baculovirus transfer vector to facilitate transfer to AcMNPV genome, and the recombinant baculovirus was used to transfect Sf9 cells. Expression of MIP was confirmed by immunoblotting and purified by HPLC. Reconstituted liposomes were used to study the function of the recombinant protein.
Results:
SDS-PAGE and immunoblots confirmed that the expressed protein was targeted to the cell membrane. MIP accounted for about 10% of total membrane proteins of the transfected Sf9 cell membranes. Molecular size 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 studies showed that the channels formed by recombinant MIP are functionally similar to those of native MIP isolated from calf lens membranes.
Conclusions:
This study describes functional expression of MIP in a baculovirus expression system. This opens the way to studying the role of MIP and to understanding the effect of mutations of critical residues in the function of MIP.