Secretion of human epidermal growth factor from Saccharomyces cerevisiae using synthetic leader sequences.
We have investigated different leader sequences for their ability to direct the efficient secretion of human epidermal growth factor (hEGF) from Saccharomyces cerevisiae. We designed a consensus signal sequence which directs secretion of hEGF from yeast as efficiently as the yeast invertase signal sequence. However, secretion is increased over fivefold by the introduction, after the signal sequence, of a synthetic 19-amino acid (aa) pro-sequence containing a cleavage recognition site for the KEX2 protease. Even in the absence of an Asn-linked glycosylation site, secretion of hEGF using the synthetic prepro-leader was as efficient as that directed by the alpha-factor leader. The role of the KEX2 protease cleavage site was investigated by mutation of the yeast alpha-factor KEX2 site (cleavage after Lys-Arg). Cleavage was obtained with the following order of efficiency, Lys-Arg greater than Pro-Arg greater than Asp-Arg, although the sequence context was also found to affect efficiency.
British Bio-technology Ltd., Cowley, Oxford, U.K.
J M
JM
Clements
G H
GH
Catlin
M J
MJ
Price
R M
RM
Edwards
D000595:Amino Acid Sequence; D001483:Base Sequence; D016384:Consensus Sequence / Q000235:genetics / Q000502:physiology; D004252:DNA Mutational Analysis; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic / Q000235:genetics / Q000502:physiology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D000072235:Mating Factor; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D043484:Proprotein Convertases; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000378:metabolism; D013381:Subtilisins; D043324:beta-Fructofuranosidase
D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D004815:Epidermal Growth Factor; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae