Characterization of proteinase A glycoforms from recombinant Saccharomyces cerevisiae.
Proteinase A produced by Saccharomyces cerevisiae carrying the gene PEP4 (which encodes the prepro-proteinase A on multicopy plasmids) was isolated and characterized by means of SDS/PAGE, laser desorption m.s. (l.d.m.s.) and high-performance capillary electrophoresis. Although proteinase A is normally located in the yeast vacuole, overexpression resulted in it being secreted. SDS/PAGE revealed that the product isolated from the culture medium comprised an approx, 7:3 mixture of two different forms of proteinase A, the apparent molecular masses of which were 42 and 40 kDa respectively. The exact mass of each form, measured by l.d.m.s., was 40,755 and 38,132 Da respectively. Further analysis employing N-glycosidase F digestion and CNBr cleavage revealed that the larger molecule was native proteinase A bearing carbohydrate moieties at Asn68 and Asn269, whereas the smaller molecule was a proteinase A variant glycoform lacking the carbohydrate moiety at Asn269. Capillary electrophoresis of both the normal and underglycosylated proteinase A glycoforms revealed charge heterogeneities attributable to differences in the phosphorylation level of the carbohydrate moiety at Asn68.
Pedersen
J
J
Department of Biotechnology, Technical University of Denmark, Lyngby.
Biedermann
K
K
D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D002241:Carbohydrates; D002455:Cell Division; D003470:Culture Media; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D013058:Mass Spectrometry; D008970:Molecular Weight; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D002241:Carbohydrates; D003470:Culture Media; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006026:Glycoside Hydrolases; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases; D003488:Cyanogen Bromide