Structure-function relationships of the erythropoietin molecule.
The tertiary structure of erythropoietin (EPO) remains to be elucidated by X-ray crystallography. Although the amino acid sequence of EPO is known, the specific features that confer its biological activity are not well understood. In order to study the structure-function relationships of EPO by in vitro mutagenesis, we have used the vector pGEX-2T to express human and murine EPO fused to the carboxyl terminus of glutathione S-transferase (GST) in E. coli. The fusion proteins were the predicted size (46 kDa) by SDS-PAGE. GST-huEPO eluted from glutathione-agarose using reduced glutathione (GSH) was tested by radioimmunoassay and in a mouse spleen cell assay (MSCA). Dose-response curves parallel to recombinant human EPO (rHuEPO) were obtained in both assays. The ratio of immuno- to bioactivity was 4.7:1. Thus the presence of the 26 kDa GST protein at the end terminus of EPO does not abrogate biological activity. GST-mEPO also gave dose-response curves parallel to rHuEPO in the MSCA but not in the RIA. The wild-type murine and three mutant GST-EPO fusion proteins (166 Des-Arg, Glu 159-->Val, and Arg 163-->Glu) were tested in the MSCA and assayed for GST activity. The ratio of bioactivity to enzyme activity for the Arg 163-->Glu mutant was approximately one third of the value obtained for each of the other fusion proteins, indicating that arginine at 163 is functionally important for EPO activity. The availability of these human and murine gene constructs in pGEX should facilitate site-directed mutagenesis and permit detailed studies of the structure-function relationships for the two erythropoietins.
Lappin
T R
TR
Department of Haematology, Royal Victoria Hospital, Belfast, Northern Ireland.
Winter
P C
PC
Elder
G E
GE
McHale
C M
CM
Hodges
V H
VH
Bridges
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002520:Cercopithecidae; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D004921:Erythropoietin / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D015870:Gene Expression; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D005982:Glutathione Transferase