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.
McAleer
B
B
School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, United Kingdom. barry.mcaleer@aht.org.uk
Rima
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009109:Mumps virus / Q000235:genetics*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D013696:Temperature; D014170:Transformation, Genetic; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000378:metabolism
D014760:Viral Fusion Proteins; D000432:Methanol