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 alpha-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.