We used a variant of GIRK2 shown previously to express efficiently in Pichia pastoris; mouse GIRK2 (containing amino acids 52–380) is linked in-frame with an HRV 3 C protease site, green fluorescent protein (GFP) and a decahistidine (HIS10) tag (a generous gift from R. MacKinnon, The Rockefeller University, New York, NY). GIRK2 clones in pPICZ were transformed into Pichia pastoris using the lithium chloride transformation method70 or electroporation (according to manufacturer protocols). Transformants were screened based upon Zeocin resistance (>500 μg/ml) and GFP emission. Highest expressing clones were selected for large-scale purification.
