Expression and mutagenesis of recombinant human and murine erythropoietins in Escherichia coli.
Expression of the polypeptide hormone erythropoietin (EPO) in Escherichia coli by four bacterial expression vectors was examined. Complementary DNAs encoding human and murine EPO were amplified by polymerase chain reaction (PCR) and cloned into the glutathione-S-transferase (GST) fusion vector, pGEX-2T. Human EPO DNA was also cloned into the vectors, pET14b, pIN III-Omp A2 and pT7/7. Expression of human and murine EPO was obtained using constructs based on pGEX-2T. For constructs based on the other vectors, expression of EPO was absent or occurred at low levels, despite attempts to optimise conditions. Human and murine EPO, expressed as fusion proteins with GST, were partially soluble and displayed EPO bioactivity. Soluble GST-EPO fusion proteins were affinity purified on immobilised glutathione. Insoluble protein could also be purified by elution from gel slices following SDS-PAGE to yield either fusion protein or, after treatment with thrombin, unmodified EPO which was both soluble and bioactive. The pGEX expression system was evaluated as a means of analysing the structure-function relationships of EPO by in vitro mutagenesis. Three human and three murine EPO mutants were constructed and expressed as GST fusion proteins. Following purification, biological activity was evaluated using assays for bioactivity, immunoactivity and GST activity. The pGEX expression system complements eukaryotic systems described previously for expression of EPO and should provide much useful information about the structure-function relationships of the hormone.
Bill
R M
RM
Dyson Perrins Laboratory, University of Oxford, UK.
Winter
P C
PC
McHale
C M
CM
Hodges
V M
VM
Elder
G E
GE
Caley
J
J
Flitsch
S L
SL
Bicknell
R
R
Lappin
T R
TR
D000740:Anemia / Q000188:drug therapy; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000235:genetics; D004920:Erythropoiesis / Q000187:drug effects; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility; D013045:Species Specificity; D013154:Spleen / Q000187:drug effects; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D005982:Glutathione Transferase; D013917:Thrombin; D005978:Glutathione