Purification of porcine phospholamban expressed in Escherichia coli.
Phospholamban (PLB) is a small hydrophobic protein that regulates contractility in the heart. This membrane protein expressed in bacterial cells 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 the PLB gene by the polymerase chain reaction from genomic DNA of porcine 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 protein was found associated predominantly with insoluble cellular constituents. However, most of the fusion protein was readily extracted with SDS. PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS-gel electrophoresis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin. This isolation method yields 3-4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteoliposomes.
Yao
Q
Q
Department of Biochemistry, University of Kansas, Lawrence 66045, USA.
Bevan
J L
JL
Weaver
R F
RF
Bigelow
D J
DJ
D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013552:Swine; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D011993:Recombinant Fusion Proteins; D000099211:Phospholamban