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Related Experiment Videos

Covalent dimerization of recombinant human interferon-gamma

S L Lauren1, T Arakawa, K Stoney

  • 1Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1789.

Archives of Biochemistry and Biophysics
|November 1, 1993
PubMed
Summary

Recombinant human interferon-gamma undergoes C-terminal processing and covalent dimerization during storage, forming a novel peptide bond between processed and intact protein chains.

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Area of Science:

  • Biochemistry
  • Protein Chemistry
  • Molecular Biology

Background:

  • Recombinant human interferon-gamma (rhIFN-γ) is a therapeutic protein.
  • Consistent observation of a nonreducible covalent dimer via SDS-PAGE necessitates investigation.

Purpose of the Study:

  • To elucidate the nature of covalent dimerization in stored rhIFN-γ.
  • To identify the molecular mechanisms underlying dimer formation and C-terminal processing.

Main Methods:

  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions.
  • Gel filtration chromatography in 8 M urea.
  • Reverse-phase chromatography.
  • Mass spectrometry (MS).
  • Amino acid sequencing.

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  • Tryptic peptide mapping.

    • Main Results:

    • Stored rhIFN-γ exhibited C-terminal processing (loss of 7 amino acids) and covalent dimerization.
    • Mass spectral analysis identified monomeric (16,900 Da, 16,100 Da) and dimeric (33,000 Da) species.
    • Tryptic peptide mapping revealed a unique peptide linking C-terminal residues (133-137) to N-terminal residues (1-7) of another molecule.

    Conclusions:

    • Recombinant methionyl human interferon-gamma undergoes specific C-terminal cleavage after phenylalanine 137.
    • A peptide bond forms between residue 137 and the N-terminal methionine (residue 1) of another molecule, creating the observed dimer.
    • Both C-terminal processing and dimerization occur during protein storage.