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

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

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