Expression, glycosylation and secretion of yeast acid phosphatase in hamster BHK cells

The gene PHO5 coding for one of the repressible acid phosphatases of the yeast Saccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human beta-actin promoter and was transfected into B...

Full description

Saved in:
Bibliographic Details
Published in:Glycoconjugate journal 1992-02, Vol.9 (1), p.39-44
Main Authors: RELJIC, R, BARBARIC, S, RIES, B, BUXTON, R, COLIN HUGHES, R
Format: Article
Language:English
Subjects:
Acid Phosphatase - biosynthesis
Acid Phosphatase - physiology
Acid Phosphatase - secretion
Analytical, structural and metabolic biochemistry
Animals
Base Sequence
Biological and medical sciences
Cell Line
Cricetinae
Electrophoresis, Polyacrylamide Gel
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Genetic Vectors - genetics
Glycopeptides - analysis
Glycosylation
Hydrolases
Molecular Sequence Data
Saccharomyces cerevisiae - enzymology
Transformation, Genetic - genetics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The gene PHO5 coding for one of the repressible acid phosphatases of the yeast Saccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human beta-actin promoter and was transfected into BHK cells by the calcium phosphate method. The recombinant APase (r-APase) which was secreted in active form from the cells was estimated by SDS/polyacrylamide gel electrophoresis to have molecular mass M(r) = 62,000, indicating substitution of the polypeptide moiety by 2-3 asparagine-linked glycans. Analysis by sequential lectin affinity chromatography of glycopeptides obtained from r-APase with Pronase showed that the glycans are predominantly of the 2.2.4 triantennary and tetraantennary complex-type. These data suggest that the extensive glycosylation of yeast APase, which contains eight polymannose substituents, is not essential for secretion and expression of enzymatic activity of the transfected gene product.
ISSN:0282-0080
1573-4986
DOI:10.1007/BF00731176