Characterization of the isoforms of PIXY321, a granulocyte-macrophage-colony stimulating factor-interleukin-3 fusion protein, separated by preparative isoelectric focusing on immobilized pH gradients

We present here the purification and the characterization of the isoforms o f PIXY321, a genetically engineered fusion of granulocyte-macrophage-colony stimulating factor and interleukin-3 expressed in yeast. The isoforms of P IXY321 were isolated using preparative isoelectric focusing (IEF) on immobi lized pH gradients. Analysis of the collected fractions on analytical IEF g els showed that PIXY321 was resolved into four discrete isoforms of isoelec tric point (pI) 5.0, 5.1, 5.2 and 5.3 with excellent yields. Subsequent ana lysis of purified isoforms of PIXY321 by peptide mapping and mass spectrome try linked the microheterogeneity of the original molecule to three paramet ers, the presence of deamidated residues, charged glycans rind the pattern of O-linked glycosylation along the peptide sequence. This last parameter e mphasizes the role of conformational aspects as key factors influencing the apparent isoelectric point of protein isoforms. (C) 1999 Elsevier Science B.V. All rights reserved.