The Use of Antibodies for Characterization and Quantification of a Recombinant Protein

In this study, we characterized proteinase A secreted by recombinant Saccharomyces cerevisiae bearing a multicopy plasmid containing the encoding gene (PEP4). Polyclonal and monoclonal antibodies were raised to study the product heterogeneity. Characterization of proteinase A was performed by immuno...

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Bibliographic Details
Published in:Annals of the New York Academy of Sciences 1996-05, Vol.782 (1), p.462-477
Main Authors: SABATER, MARGARITA, HEILMANN, SUSANNE, FRØKIÆR, HANNE, BIEDERMANN, KIRSTEN, EMBORG, CLAUS
Format: Article
Language:English
Subjects:
Animals
Antibodies
Antibodies, Monoclonal
Antibody Specificity
Aspartic Acid Endopeptidases - analysis
Aspartic Acid Endopeptidases - biosynthesis
Aspartic Acid Endopeptidases - chemistry
Cloning, Molecular - methods
Cross Reactions
Cyanogen Bromide
Electrophoresis, Agar Gel
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Genes, Fungal
Glycosylation
Immunoblotting
Immunoelectrophoresis, Two-Dimensional
Isoelectric Focusing
Mice
Peptide Fragments - isolation & purification
Plasmids
Rabbits
Recombinant Proteins - analysis
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Saccharomyces cerevisiae
Schizosaccharomyces - enzymology
Schizosaccharomyces - genetics
Triose-Phosphate Isomerase - analysis
Triose-Phosphate Isomerase - biosynthesis
Triose-Phosphate Isomerase - chemistry
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Summary:In this study, we characterized proteinase A secreted by recombinant Saccharomyces cerevisiae bearing a multicopy plasmid containing the encoding gene (PEP4). Polyclonal and monoclonal antibodies were raised to study the product heterogeneity. Characterization of proteinase A was performed by immunoelectrophoresis and immunoblotting techniques. None of the monoclonal antibodies raised against proteinase A was found to react with the glycosyl side chains; thus cross-reaction with other glycosylated proteins (e.g. carboxypeptidase Y) was very low. This study allowed us to develop an ELISA method for the quantification of proteinase A in culture supernatants as well as the evaluation of monoclonal antibodies for their use in immunoaffinity chromatography.
ISSN:0077-8923
1749-6632
DOI:10.1111/j.1749-6632.1996.tb40584.x