Alpha-factor-leader-directed secretion of recombinant human-insulin-like growth factor I from Saccharomyces cerevisiae. Precursor formation and processing in the yeast secretory pathway.
A synthetic gene coding for human-insulin-like growth factor I (IGFI) was fused to the leader sequence of yeast prepro-alpha-factor and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter fragment. Recombinant IGFI was found inside yeast cells and secreted into the medium. The secreted IGFI migrated on SDS gels with the same electrophoretic mobility as authentic IGFI, i.e. at about 7.5 kDa. HPLC analysis of secreted IGFI revealed the presence of the correctly folded, genuine molecule as well as an isomeric byproduct of equal molecular mass but with two of the three disulfide bonds interchanged. Inside exponentially growing cells the 7.5-kDa IGFI was also found, along with up to four additional IGFI-related polypeptides of higher molecular mass. By endoglycosidase F treatment the three polypeptides between 19-26 kDa were converted to a single peptide of 17 kDa. Since this peptide also reacted with an anti-alpha-factor antibody, it represents most likely the unglycosylated alpha-factor--IGFI fusion precursor. Pulse-chase experiments established the precursor nature of the intracellular higher-molecular-mass IGFI species. Conversion of the primary translation product to the differently glycosylated IGFI precursor proteins and into the mature form occurred very rapidly, within 2 min. Rapid maturation was, however, not followed by an equally rapid secretion of the mature form into the medium: only after 30-40 min did IGFI appear outside the cells. We therefore postulate the presence of an as yet undefined Golgi or post-Golgi bottleneck representing a major obstacle in secretion of recombinant IGFI from S. cerevisiae cells.
Steube
K
K
Biotechnology Department Ciba, Geigy Ltd, Basel, Switzerland.
Chaudhuri
B
B
Märki
W
W
Merryweather
J P
JP
Heim
J
J
D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005656:Fungal Proteins / Q000235:genetics*; D014644:Genetic Variation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013104:Spheroplasts / Q000378:metabolism
D005656:Fungal Proteins; C048632:MF(ALPHA)1 protein, S cerevisiae; D011498:Protein Precursors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D007334:Insulin-Like Growth Factor I; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases