Separation of gamma-globulins from porcine plasma by reversed micellar extraction.

Factors affecting the separation of gamma-globulins from porcine plasma using reversed micelles were screened based on a fractional factorial design. The optimal processing conditions for obtaining the maximum yield of gamma-globulins under various constraints of product purity were determined using response surface methodology (RSM) and nonlinear programming. Results showed that the pH and sodium chloride concentration of the aqueous phase, and the concentration of surfactant (bis-(2-ethylhexyl) sulfosuccinate sodium salt, AOT) of the organic phase were the most important factors affecting the extraction performance.

The extracytoplasmic domain of the erythropoietin receptor forms a monomeric complex with erythropoietin.

We have generated a truncated form of the erythropoietin receptor (EPO-R), the extracytoplasmic ligand-binding domain, that is secreted from a transfected Chinese hamster ovary (CHO) cell line. The truncated receptor is readily purified from CHO conditioned media as a 33-Kd glycosylated protein, which is converted to a 25-Kd species upon treatment with protein N-glycan glycosidase F. Cross-linking of radioiodinated EPO to the secreted receptor yielded a complex of 72 Kd.

The distribution of glycan structures in individual N-glycosylation sites in animal and plant glycoproteins.

Glycopeptides representing each individual N-glycosylation site in six animal and plant glycoproteins (ovoinhibitor and ovotransferrin, orosomucoid, antitrypsin, phaseolin, and phytohemagglutinin) have been isolated and compared by mass spectrometric analysis. Since the isolation step separates each individual peptide regardless of the nature of the glycan attached to it, it is possible to observe the entire spectrum of glycans associated with each site from the mass spectrum of the corresponding glycopeptide. The three glycosylation sites in ovoinhibitor have very similar but not identical glycans; they are significantly different from those observed in the single site of ovotransferrin.

New methods for rapid separation and detection of oligosaccharides from glycoproteins.

Ion exchange chromatography at high pH with pulsed amperometric detection of the eluted glycans permitted resolution of the eight major components in the mixture of asparagine-linked glycans derived from the single glycosylation site of ovalbumin. The individual glycans were first partially separated according to size, and were characterized by fast atom bombardment-mass spectrometry and specific enzymatic degradation with beta-galactosidase and endoglycosidase H; subnanomolar quantities of all eight components could subsequently be unequivocally identified in the elution diagram. To ascertain that the chromatographic separation of the ovalbumin glycan mixture was not restricted to the asparagine-linked glycans, it was established that the corresponding mixture of reducing oligosaccharides (asparagine removed) or Asn-oligosaccharides blocked at the alpha-amino group with biotin gave very similar resolution of the eight glycans.

Purification and characterization of two glycopeptide hydrolases from jack beans.

Two glycopeptide hydrolases, an endo-beta-N-acetylglucosaminidase and peptide:N-glycanase (amidase), have been isolated from defatted jack bean meal by standard procedures involving differential solubility and column chromatography. The purified products appear to be free of contaminating proteases and exoglycosidases, and their substrate specificity has been explored with regard to both glycan and peptide structure of the substrates. The endoglycosidase appears to be specific for high mannose glycans; no hydrolysis of either hybrid or complex glycans has been observed.