Refolding, purification, and characterization of human erythropoietin binding protein produced in Escherichia coli

Protein Expression and Purification
D L JohnsonL K Jolliffe

Abstract

The extracellular domain of the human erythropoietin receptor (EPO binding protein (EBP)) has been expressed and overproduced in Escherichia coli. Regardless of the presence ofpelB or ompT signal sequences the recombinant protein produced in this fashion appears, as with many other recombinant eukaryotic proteins produced in E. coli as an insoluble product in laboratory scale fermentations. The induction product of the pelB protein expression system appears as two protein forms with slightly different molecular weights. Based on N-terminal sequence analysis of recovered protein, these forms represent two variants, one with the signal sequence properly processed to yield the expected "native" amino terminus and another which retains the signal sequence. Both forms appear as insoluble fermentation products. Control of oxygen levels and pH during high density fermentation allows the production of only the protein variant with the native amino terminus. Methods reported here permit the efficient recovery of purified EBP which quantitatively binds EPO in solution as determined by high performance size exclusion chromatography. A long-lived refolding intermediate was observed which penultimately collapses into an active conformation....Continue Reading

Citations

Oct 16, 1999·Proceedings of the National Academy of Sciences of the United States of America·S A QureshiD F Mark
Mar 12, 2013·Cellular and Molecular Life Sciences : CMLS·Samuel S NewtonRonald S Duman
Oct 13, 2004·Protein Expression and Purification·Amardeep KhushooK J Mukherjee
Feb 21, 1997·The Journal of Biological Chemistry·F P BarboneL S Mulcahy
Jul 9, 2008·The Journal of Immunology : Official Journal of the American Association of Immunologists·Susan E LacyEdward B Reilly
Sep 16, 2021·Angewandte Chemie·Hendrik HessefortCarlo Unverzagt

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