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 of PIXY321, a genetically engineered fusion of granulocyte-macrophage-colony stimulating factor and interleukin-3 expressed in yeast. The isoforms of PIXY321 were isolated using preparative isoelectric focusing (IEF) on immobilized pH gradients. Analysis of the collected fractions on analytical IEF gels showed that PIXY321 was resolved into four discrete isoforms of isoelectric point (pI) 5.0, 5.1, 5.2 and 5.3 with excellent yields. Subsequent analysis of purified isoforms of PIXY321 by peptide mapping and mass spectrometry linked the microheterogeneity of the original molecule to three parameters, the presence of deamidated residues, charged glycans and the pattern of O-linked glycosylation along the peptide sequence. This last parameter emphasizes the role of conformational aspects as key factors influencing the apparent isoelectric point of protein isoforms.
Balland
A
A
Immunex Corp., Seattle, WA 98101, USA.
Mahan-Boyce
J A
JA
Krasts
D A
DA
Daniels
M
M
Wang
W
W
Gombotz
W R
WR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007377:Interleukin-3 / Q000737:chemistry* / Q000302:isolation & purification; D007525:Isoelectric Focusing / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry; D020033:Protein Isoforms / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification
D000596:Amino Acids; D007377:Interleukin-3; C078524:PIXY321 fusion protein, recombinant; D011134:Polysaccharides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor