A removable spacer peptide in an alpha-factor-leader/insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae.
An alpha-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).
Kjeldsen
T
T
Molecular Biology and Protein Chemistry, Novo Nordisk A/S, Bagsvaerd, Denmark.
Brandt
J
J
Andersen
A S
AS
Egel-Mitani
M
M
Hach
M
M
Pettersson
A F
AF
Vad
K
K
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000378:metabolism*
D005656:Fungal Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D011384:Proinsulin; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; C499266:YPS1 protein, S cerevisiae