COS-1 cell expression and one-step affinity protein purification and activity of epitope-tagged human erythropoietin and of site-directed mutants.
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.
Bill
R M
RM
The Dyson Perrins Laboratory, Oxford, UK.
Flitsch
S L
SL
Bicknell
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells / Q000378:metabolism*; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000302:isolation & purification; D004921:Erythropoietin / Q000235:genetics* / Q000302:isolation & purification*; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D014162:Transfection
D018076:DNA, Complementary; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin