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Abstract

Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin.

Hangjun Zhan, Beishun Liu, Scott W Reid, Kennneth H Aoki,Cuiwei Li, Rashid S Syed, Cyrus Karkaria, Gary Koe, Karen Sitney, Kirk Hayenga, Firoz Mistry, Laura Savel, Mark Dreyer, Bradley A Katz, Jolanda Schreurs, David J Matthews, Janet C Chreetham, Joan Egrie, Lutz B Giebel and Robert M Stroud

Protein Engineering 12, 505-513, June 1999
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The extracellular ligand-binding domain (EPObp) of the human EPO receptor (EPOR) was expressed  both in CHO (Chinese Hampster Ovary) cells and in Pichia pastoris.  The CHO and yeast expressed receptors showed identical affinity for EPObinding.  Expression levels in P. pastoris were significantly higher, favoring its use as an expression and scale-up production system. Incubation of EPO with a four-fold molar excess of receptor at high protein concentrations yielded stable EPO-EPObp complexes.  Quantification of EPO and EPObp in the complex yielded a molar ratio of one EPO molecule to two receptor molecules. 

Residues that are responsible for EPOR glycosylation and isomerization in Pichia were identified and eliminated by site-specific mutagenesis.  A thiol modification was identified and a method was developed to remove the modified species from EPObp.  EPObp was complexed witherythropoietin (EPO) and purified.  The complex crystallized in two crystal forms that diffracted to 2.8 and 1.9 Å respectively.  (Form 1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals, Inc. and Amgen, Inc respectively.)  Both contained one complex per asymmetric unit with a stoichiometry of two EPObps to one EPO.

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