Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin.
The extracellular ligand-binding domain (EPObp) of the human EPO receptor (EPOR) was expressed both in CHO (Chinese Hamster Ovary) cells and in Pichia pastoris. The CHO and yeast expressed receptors showed identical affinity for EPO binding. Expression levels in P. pastoris were significantly higher, favoring its use as an expression and scale-up production system. Incubation of EPO with a fourfold 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 with erythropoietin (EPO) and purified. The complex crystallized in two crystal forms that diffracted to 2.8 and 1.9 A 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.
Zhan
H
H
Axys Pharmaceuticals, Inc., 180 Kimball Way, San Francisco, CA 94080, USA.
Liu
B
B
Reid
S W
SW
Aoki
K H
KH
Li
C
C
Syed
R S
RS
Karkaria
C
C
Koe
G
G
Sitney
K
K
Hayenga
K
K
Mistry
F
F
Savel
L
L
Dreyer
M
M
Katz
B A
BA
Schreurs
J
J
Matthews
D J
DJ
Cheetham
J C
JC
Egrie
J
J
Giebel
L B
LB
Stroud
R M
RM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003460:Crystallization; D003545:Cysteine / Q000032:analysis; D004921:Erythropoietin / Q000737:chemistry*; D015870:Gene Expression; D005978:Glutathione / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D014961:X-Ray Diffraction
D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D005978:Glutathione; D003545:Cysteine