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.

• Publication year

  1993

• Venue

  Biotechnology and applied biochemistry

• Publication date

  1993-12-01

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1111/J.1470-8744.1993.TB00278.XPMID 8297512
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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