Premium
Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients
Author(s) -
Bischoff Rainer,
Roecklin Dominique,
Roitsch Carolyn
Publication year - 1992
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150130144
Subject(s) - isoelectric focusing , isoelectric point , chromatography , recombinant dna , chemistry , affinity chromatography , cysteine , amino acid , biochemistry , agarose , ion chromatography , iodoacetamide , hydrophilic interaction chromatography , high performance liquid chromatography , enzyme , gene
Isoelectric focusing in immobilized pH gradients (IEF‐IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion‐exchange and reversed‐phase chromatography proved to be homogeneous with regard to its isoelectric point (p I ). In addition, the theoretical p I , calculated on the basis of the primary structure, corresponded precisely to the measured p I of 4.30. IEF‐IPG was further employed to follow the stability of recombinant hirudin at pH 9, indicating that deamidation occurred under these conditions. A variant of recombinant human α 1 ‐antitrypsin (AAT) (389 amino acids, one cysteine residue) expressed in Escherichia coli and purified by anion‐exchange, metal chelate and hydrophobic‐interaction chromatography appeared to be homogeneous by polyacrylamide gel electrophoresis under reducing and denaturing conditions as well as by various high performance liquid chromatography methods. However, some heterogeneity was detected by IEF‐IPG between pH 5–6. The measured p I values of 5.43–5.58 were slightly lower than the calculated p I based on the primary structure (5.72). This indicated deamidations of Asn or Gln residues. A recombinant Schistosoma mansoni parasite antigen, p28 (210 amino acids, one cysteine residue) obtained after intracellular expression in Saccharomyces cerevisiae and affinity purification on glutathione agarose was analyzed by IEF‐IPG in a pH 7.3–8.3 gradient. It appeared to be heterogeneous with regard to its p I , with the major component having a p I of 7.81 compared to the calculated value of 7.17. N ‐Terminal amino acid sequencing as well as amino acid composition analysis were performed on the separated forms of p28 after electroblotting showing the feasibility of combining IEF‐IPG with subsequent analytical methods to obtain structural information.