COS-1 cell expression and one-step affinity protein purification and activity of epitope-tagged human erythropoietin and of site-directed mutants

Roslyn M Bill, Sabine L Flitsch, Roy Bicknell

Research output: Contribution to journalArticle

Abstract

Recombinant human erythropoietin (rhEPO) is an important glycoprotein hormone which has been successfully used in the treatment of anaemia. To facilitate the rapid evaluation of wild-type and mutant forms of rhEPO in structure-function studies, we have developed an expression system in which the recombinant hormone is tagged at the C-terminus with a c-myc peptide. One-step affinity purification of culture supernatants on an anti-myc antibody column yielded proteins which were greater than 50% pure with a specific activity of 300,000 U/mg, in agreement with the value of wild-type protein. We conclude that the additional myc-peptide does not affect receptor binding. The expression system was used to study three mutants in which the N-glycosylation sites were changed to cysteines (Asn24Cys, Asn38Cys and Asn83Cys). Specific activities of these cysteine mutants were significant, but reduced (60%, 22% and 70%, respectively), compared to wild-type. The reduction in specific activity may be due to reduced stability of the mutant proteins.
Original languageEnglish
Pages (from-to)13-20
Number of pages8
JournalBBA - Reviews on Biomembranes
Volume1340
Issue number1
DOIs
Publication statusPublished - 20 Jun 1997

Fingerprint

COS Cells
Erythropoietin
Cysteine
Epitopes
Hormones
Peptides
Mutant Proteins
Glycosylation
Anemia
Anti-Idiotypic Antibodies
Glycoproteins
Proteins

Keywords

  • A mino Acid Sequence
  • Animals
  • Base Sequence
  • COS Cells
  • Chromatography, Affinity
  • DNA, Complementary
  • Erythropoietin
  • Gene Expression
  • Genetic Vectors
  • Glycosylation
  • Humans
  • Mice
  • Mutagenesis, Site-Directed
  • Proto-Oncogene Proteins c-myc
  • Recombinant Fusion Proteins
  • Transfection

Cite this

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title = "COS-1 cell expression and one-step affinity protein purification and activity of epitope-tagged human erythropoietin and of site-directed mutants",
abstract = "Recombinant human erythropoietin (rhEPO) is an important glycoprotein hormone which has been successfully used in the treatment of anaemia. To facilitate the rapid evaluation of wild-type and mutant forms of rhEPO in structure-function studies, we have developed an expression system in which the recombinant hormone is tagged at the C-terminus with a c-myc peptide. One-step affinity purification of culture supernatants on an anti-myc antibody column yielded proteins which were greater than 50{\%} pure with a specific activity of 300,000 U/mg, in agreement with the value of wild-type protein. We conclude that the additional myc-peptide does not affect receptor binding. The expression system was used to study three mutants in which the N-glycosylation sites were changed to cysteines (Asn24Cys, Asn38Cys and Asn83Cys). Specific activities of these cysteine mutants were significant, but reduced (60{\%}, 22{\%} and 70{\%}, respectively), compared to wild-type. The reduction in specific activity may be due to reduced stability of the mutant proteins.",
keywords = "A mino Acid Sequence, Animals, Base Sequence, COS Cells, Chromatography, Affinity, DNA, Complementary, Erythropoietin, Gene Expression, Genetic Vectors, Glycosylation, Humans, Mice, Mutagenesis, Site-Directed, Proto-Oncogene Proteins c-myc, Recombinant Fusion Proteins, Transfection",
author = "Bill, {Roslyn M} and Flitsch, {Sabine L} and Roy Bicknell",
year = "1997",
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journal = "BBA - Reviews on Biomembranes",
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COS-1 cell expression and one-step affinity protein purification and activity of epitope-tagged human erythropoietin and of site-directed mutants. / Bill, Roslyn M; Flitsch, Sabine L; Bicknell, Roy.

In: BBA - Reviews on Biomembranes, Vol. 1340, No. 1, 20.06.1997, p. 13-20.

Research output: Contribution to journalArticle

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AU - Bill, Roslyn M

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KW - Gene Expression

KW - Genetic Vectors

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KW - Mice

KW - Mutagenesis, Site-Directed

KW - Proto-Oncogene Proteins c-myc

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KW - Transfection

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