Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant Escherichia coli.
Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S-transferase (GST). GST-PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity-purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.
Krömer
W J
WJ
Institute for Biotechnology, ETH Zürich, Switzerland.
Carafoli
E
E
Bailey
J E
JE
D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D019281:Dimerization; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D009206:Myocardium / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012519:Sarcoplasmic Reticulum / Q000737:chemistry; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D004765:Enteropeptidase; D005982:Glutathione Transferase; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D000099211:Phospholamban