An attempt was made to make protein bands visible on native gel using copper staining, since such a mild staining procedure would make the entire native gel electrophoresis process non-denaturing. Copper staining not only was able to detect various proteins on native gel with reasonable sensitivity, but also made extraction and recovery of active proteins possible from the gel using a gentle procedure.

Human erythropoietin (EPO) produced in Chinese hamster ovary cells (CHO-EPO) is a hydrophobic protein stabilized by the highly branched complex-type N-glycans. To characterize the stabilizing effect of the N-glycans, the properties of enzymatically N-glycan-modified CHO-EPO species were compared spectrophotometrically. CD and fluorescence spectra following the protein unfolding induced by guanidine hydrochloride or pH revealed that the inner regions including the galactose residues of the N-glycans stabilize the protein conformation. The decrease in the conformational stability caused by enzymatic trimming of the N-glycans was associated with the exposure of the hydrophobic protein surface areas accessible to 1-anilino-8-naphthalenesulfonic acid (ANS) binding. Further, the ANS binding and heat denaturation of Escherichia coli-expressed EPO (nonglycosylated EPO) were depressed in dilute solutions (1 mM or so) of free N-glycans of the complex type. These results, together with the finding that the N-glycans of CHO-EPO make little contact with the aromatic amino acid residues exposed on the protein surface, indicate that the inner regions including the galactose residues of the intramolecular N-glycans stabilize the protein conformation by clinging to the hydrophobic protein surface areas mainly made up of nonaromatic hydrocarbon groups.

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.

Although there is considerable evidence that signaling by the erythropoietin (EPO) receptor is initiated when it is dimerized by binding EPO, it has been previously reported that the soluble extracellular domains of the EPO receptor (sEPOR) are not dimerized in the presence of EPO and are able to form only 1:1 complexes with EPO. We have now shown unambiguously by light scattering, sedimentation equilibrium, and titration calorimetry that two molecules of sEPOR can bind to a single EPO monomer but that the binding of the second sEPOR is approximately 1000-fold weaker than that of the first. Because this second binding interaction is quite weak (Kd of approximately 1 microM), the 2:1 sEPOR.EPO complexes are easily dissociated during chromatography (forming the 1:1 complexes reported previously) and cannot be isolated in pure form. Global analysis of the sedimentation equilibrium data has enabled us to determine the binding constants and is consistent with a model in which EPO has two independent binding sites for sEPOR but cannot exclude anticooperative or sequential binding models. The influence of glycosylation of EPO and/or sEPOR on the binding affinities has also been investigated. Titration calorimetry is consistent with the sedimentation data and shows that the weaker binding site has a more negative delta H. The relation of these results to the binding of EPO to membrane-bound receptors and to the phenomenon of apparent high-affinity and low-affinity classes of receptors is discussed.

Erythropoietin is a glycoprotein hormone that stimulates the maturation of late erythroid progenitor cells. It has three N-linked and one O-linked carbohydrates which play an important role in the biosynthesis and biological activities of the protein. To determine the role the carbohydrate might have in maintaining the conformational stability of the protein, the protein expressed in mammalian cells (fully glycosylated), the asialo mammalian-expressed protein, and the protein expressed in Escherichia coli (no carbohydrate) were compared for their stability to guanidine HCl, pH, and temperature. Circular dichroism was used to follow protein unfolding. Both the intact and asialo mammalian-expressed proteins unfolded with a cooperative transition in guanidine HCl, with a midpoint at 1.75 M guanidine HCl. The E. coli-expressed material unfolded with a midpoint of 1.2 M guanidine HCl, and a delta G of unfolding which was 1.4 kcal/mol less than that of the two glycosylated molecules. The E. coli-derived protein was also significantly less stable to pH-induced conformational changes, showing a cooperative transition in 35% glycerol with a midpoint at pH 4.4, while both the intact and asialo mammalian-expressed molecules had a transition midpoint of pH 3.75 in the absence of glycerol, and approximately pH 3 in the presence of 35% glycerol. The E. coli-expressed molecule unfolded and precipitated upon heating to 44 degrees C, while the asialo and intact mammalian-expressed proteins remained soluble, with a Tm of 56 degrees C. From these experiments, the carbohydrate appears to play a critical role in stabilizing the erythropoietin molecule to denaturing conditions, and this increased stability does not depend on the presence of sialic acid.