Prepro-leaders lacking N-linked glycosylation for secretory expression in the yeast Saccharomyces cerevisiae.
Synthetic prepro-leaders lacking consensus N-linked glycosylation sites confers secretion competence of correctly folded insulin precursor expressed in the yeast species Saccharomyces cerevisiae with a yield comparable to, or better than the alpha-factor prepro-leader. In contrast, the S. cerevisiae alpha-factor prepro-leader's three N-linked oligosaccharide chains are necessary for the ability to facilitate secretion of the insulin precursor from S. cerevisiae (T. Kjeldsen et al., Biotechnol. Appl. Biochem. 27, 109-115, 1998). Synthetic prepro-leader lacking both N-glycosylation and the dibasic Kex2 endoprotease processing site also efficiently facilitated secretion of a pro-leader/insulin precursor fusion protein in which the insulin precursor was correctly folded. The unprocessed pro-leader/insulin-precursor fusion protein was purified from culture medium and matured in vitro to desB30 insulin by Achromobacter lyticus lysyl-specific protease providing an alternative yeast expression system not dependent on the Kex2 endoprotease. The synthetic prepro-leader lacking N-linked glycosylation provides the opportunity for secretory expression in yeast utilizing either in vivo Kex2 endoprotease maturation of the fusion protein during secretion or in vitro maturation of the purified fusion protein with a suitable enzyme.
Kjeldsen
T
T
Novo Nordisk A/S, Bagsvaerd, DK-2880, Denmark.
Hach
M
M
Balschmidt
P
P
Havelund
S
S
Pettersson
A F
AF
Markussen
J
J
D000421:Alcaligenes / Q000201:enzymology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D007328:Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010449:Peptide Mapping; D043484:Proprotein Convertases; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000737:chemistry / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D013381:Subtilisins / Q000502:physiology
D001426:Bacterial Proteins; D005656:Fungal Proteins; D007328:Insulin; D009844:Oligosaccharides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C036813:lysyl endopeptidase; C058751:KEX2 protein, S cerevisiae