Expression and secretion of a biologically active mouse sonic hedgehog protein by the methylotrophic yeast Pichia pastoris

We have successfully secreted the amino-terminal functional domain of mouse sonic hedgehog protein (SHH) into culture fluid using a yeast Pichia pasto ris expression system. A cDNA fragment encoding the amino-terminal domain o f mouse SHH was inserted downstream of the Saccharomyces cerevisiae alpha-m ating factor secretion signal. The DNA fragment was introduced into the hos t genome by the spheroplast transformation method. Transformants were selec ted based on their resistance to G418. His(+) transformants which showed re sistance to over 8 mg G418/ml were selected and analyzed for determination of the plasmid copy number. One His(+) clone which has eight copies of the expression cassette per genome was cultured in minimal medium deficient for histidine, and further cultured in buffered medium supplemented with metha nol which activates the AOX1 promoter. SDS-PAGE analysis indicated efficien t expression and secretion of mouse SHH into culture fluid. The yield of se creted SHH was estimated to be 50 mu g/ml. Purified protein was assayed for biological activity and found to activate the transcription of the Patched genes (Ptc-1 and Ptc-2) encoding receptors for SHH.