Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66

Protein Expression and Purification
C BinnieD I Stewart

Abstract

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.

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Citations

Mar 5, 2009·Future Microbiology·Kristof Vrancken, Jozef Anné
Apr 28, 2011·RNA Biology·Michael-Paul VockenhuberBeatrix Suess
Jun 5, 2004·Protein Expression and Purification·Yong ZhangYuan Li
Sep 26, 2020·Frontiers in Microbiology·Francesca BeriniElisa Binda
Jun 25, 2003·Protein Expression and Purification·Sally Xueying NiVadim Tsvetnitsky

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