PURIFICATION OF PORCINE PHOSPHOLAMBAN EXPRESSED IN ESCHERICHIA-COLI

Phospholamban (PLB) is a small hydrophobic protein that regulates cont ractility in the heart. This membrane protein expressed in bacterial c ells is resistant to purification by conventional strategies that have been used to isolate expressed soluble proteins. Therefore, in order to obtain both wild-type and mutant PLB proteins, we have amplified th e PLB gene by the polymerase chain reaction from genomic DNA of porcin e heart and inserted it into the pGEX-2T plasmid expression vector. In this vector, the gene product fused to glutathione S-transferase has been expressed in JM109 Escherichia coli cells. The expressed fusion p rotein was found associated predominantly with insoluble cellular cons tituents. However, most of the fusion protein was readily extracted wi th SDS. PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS-gel electrophor esis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin. This isolation method y ields 3-4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteolipos omes. (C) 1996 Academic Press, Inc.