PURIFICATION OF THE CARDIAC SARCOPLASMIC-RETICULUM MEMBRANE-PROTEIN PHOSPHOLAMBAN FROM RECOMBINANT ESCHERICHIA-COLI

Phospholamban (PLN) was expressed in Escherichia coli as a protein fus ion with glutathione S-transferase (GST). GST-PLN was mostly present i n the insoluble protein fraction and accounted for approximately 50% o f total insoluble protein. Attempts to suppress inclusion body formati on or to use GST as an affinity-purification tag failed, A successful purification method is based on preparative SDS/PAGE and electrodialys is. 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) p rotease site just in front of the PLN sequence and demonstrated the pr oteolytical liberation of PLN from the carrier protein. The approach d escribed represents a substantial advancement in PLN expression and pu rification.