Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients

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 (pI). In addition, the theoretical pI, calculated on the basis of the primary structure, corresponded precisely to the measured pI 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 alpha 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 pI values of 5.43-5.58 were slightly lower than the calculated pI 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 pI, with the major component having a pI of 7.81 compared to the calculated value of 7.17.(ABSTRACT TRUNCATED AT 250 WORDS)

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IEF-IPG analysis of recombinant leech hirudin expressed in S. cerevisiae; homogeneous pI of 4.30 matching primary structure; deamidation detected under alkaline conditions.

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