Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66.
A gene encoding the extracellular domain of the human erythropoietin receptor (EPO-R) was constructed using oligonucleotides, with a view to maintaining preferred codon usage for the Streptomycetes. The gene was subcloned into a multicopy Streptomyces-Escherichia coli shuttle vector, pCAN46 (derived from pIJ680), containing a strong constitutive promoter from the S. fradiae aph gene, a signal peptide coding region derived from the protease B gene of S. griseus, and a transcription terminator sequence also derived from the S. fradiae aph gene. Extracellular expression of authentic EPO-R by S. lividans was demonstrated using SDS-PAGE and Western blot analysis, followed by direct amino terminal sequencing of the purified product. Specific binding of S. lividans-expressed EPO-R to recombinant human glycosylated EPO was demonstrated using BIAcore (surface plasmon resonance) analysis and native gel shift assays.
Binnie
C
C
Cangene Corporation, Mississauga, Ontario, Canada. cbinnie@compuserve.com
Jenish
D
D
Cossar
D
D
Szabo
A
A
Trudeau
D
D
Krygsman
P
P
Malek
L T
LT
Stewart
D I
DI
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D017467:Receptors, Erythropoietin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013302:Streptomyces / Q000235:genetics
D003062:Codon; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins