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Darius Karkaria 《Software Testing, Verification and Reliability》1993,3(1):49-51
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Zhan Hangjun; Liu Beishan; Reid Scott W.; Aoki Kenneth H.; Li Cuiwei; Syed Rashid S.; Karkaria Cyrus; Koe Gary; Sitney Karen; Hayenga Kirk; Mistry Firoz; Savel Laura; Dreyer Mark; Katz Bradley A.; Schreurs Jolanda; Matthews David J.; Cheetham Janet C.; Egrie Joan; Giebel Lutz B.; Stroud Robert M. 《Protein engineering, design & selection : PEDS》1999,12(6):505-513
The extracellular ligand-binding domain (EPObp) of the humanEPO receptor (EPOR) was expressed both in CHO (Chinese HamsterOvary) cells and in Pichia pastoris. The CHO and yeast expressedreceptors showed identical affinity for EPO binding. Expressionlevels in P.pastoris were significantly higher, favoring itsuse as an expression and scale-up production system. Incubationof EPO with a fourfold molar excess of receptor at high proteinconcentrations yielded stable EPOEPObp complexes. Quantificationof EPO and EPObp in the complex yielded a molar ratio of oneEPO molecule to two receptor molecules. Residues that are responsiblefor EPOR glycosylation and isomerization in Pichia were identifiedand eliminated by site-specific mutagenesis. A thiol modificationwas identified and a method was developed to remove the modifiedspecies from EPObp. EPObp was complexed with erythropoietin(EPO) and purified. The complex crystallized in two crystalforms that diffracted to 2.8 and 1.9 Å respectively. (Form1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals,Inc. and Amgen, Inc. respectively.) Both contained one complexper asymmetric unit with a stoichiometry of two EPObps to oneEPO. 相似文献
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