Cloning and Secreted Expression of the Extracellular Domain of the Mumps Virus Fusion Protein in Pichia pastoris

The extracellular globular domain of the mumps virus fusion (F) protein (amino-acids 28-481) has been overexpressed from GS115 his4 Pichia pastoris cells following the generation of a recombinant clone. The heterologous protein was directed for secreted expression by in-frame cloning with the S.cerevisiae -factor secretion signal. The expressed protein was observed to secrete into the culture medium. An expressing clone was obtained initially by small-scale induction, metabolic labeling and immunoprecipitation. Expression analysis of the chosen clone was confirmed by western blotting with F protein specific polyclonal serum. The effects of culture volume, temperature and methanol concentration on the levels of expression, were studied. The results indicate that there is a balance required between the induction temperature and methanol concentration to achieve maximal expression. In addition, the presence of designated monomeric (47 K), dimeric (85–90 K) and trimeric (140 K) forms are dependent upon the induction conditions. Estimated secreted protein expression levels of > 1 mg/L were obtained in these studies. Further, the experiments demonstrate that the complete reconstruction of the KEX2 protease cleavage site is not necessary to facilitate secretion.