Sequence-specific deamidation: isolation and biochemical characterization of succinimide intermediates of recombinant hirudin.

@article{Bischoff1993SequencespecificDI,
  title={Sequence-specific deamidation: isolation and biochemical characterization of succinimide intermediates of recombinant hirudin.},
  author={Rainer Bischoff and Pierre Lepage and Michel Jaquinod and Gilles Cauet and M Acker-Klein and Daniel Clesse and M. Laporte and Alain Bayol and Alain van Dorsselaer and Carolyn A. Roitsch},
  journal={Biochemistry},
  year={1993},
  volume={32 2},
  pages={
          725-34
        }
}
Natural hirudin variant 2 with a lysine residue in position 47 (rHV2-Lys47) was produced in a genetically engineered strain of Saccharomyces cerevisiae as a secreted protein of 65 amino acids and purified to greater than 99% homogeneity. Only reversed-phase high-performance liquid chromatography (RP-HPLC) using very shallow acetonitrile gradients indicated the presence of a component in the final product (approximately 1% of total protein) with a slightly increased retention time. Using… Expand
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Recombinant hirudin variant rHV2-Lys 47 was intentionally deamidated by incubation in pH 9 phosphate buffer at 37 degrees C and acid-catalyzed carboxyl methylation enhanced, in particular, the abundance of the sequence ions in the LSIMS spectra. Expand
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Characterization of the final product by analytical HPLC, isoelectric focusing gel electrophoresis, quantitative amino acid composition and sequence analysis did not reveal any contaminants. Expand
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Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation.
TLDR
These studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively. Expand
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