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Related Experiment Videos

Recombinant-derived interleukin-1 alpha stabilized against specific deamidation.

P T Wingfield1, R J Mattaliano, H R MacDonald

  • 1Biogen SA, Geneva, Switzerland.

Protein Engineering
|October 1, 1987
PubMed
Summary

Recombinant human interleukin-1 alpha (IL-1 alpha) exists in two forms due to a modification at position 36. A mutant protein with serine at this position is more stable and suitable for studies.

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Area of Science:

  • Biochemistry
  • Protein Chemistry
  • Molecular Biology

Background:

  • Recombinant human interleukin-1 alpha (IL-1 alpha) purified from E. coli exhibits heterogeneity.
  • Isoelectric focusing reveals two main species with pI 5.45 and 5.20.

Purpose of the Study:

  • To characterize the molecular basis of IL-1 alpha heterogeneity.
  • To investigate the functional and structural implications of modifications at position 36.
  • To develop a more stable IL-1 alpha mutant for research.

Main Methods:

  • Isoelectric focusing (IEF) and chromatofocusing for protein separation.
  • N-terminal sequence analysis to identify amino acid differences.
  • Site-directed mutagenesis to create a Ser-36 mutant.
  • 1H-n.m.r. and circular dichroism for conformational analysis.

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  • Receptor binding assays.

    • Main Results:

    • The pI 5.45 species contains Asn-36, while the pI 5.20 species has Asp-36, indicating deamidation.
    • A mutant with Ser-36 is homogeneous (pI 5.45) and conformationally similar to wild-type.
    • Receptor binding affinities are identical for Asn, Asp, and Ser at position 36.
    • The Ser-36 mutant shows enhanced stability against deamidation.

    Conclusions:

    • The heterogeneity of recombinant IL-1 alpha is due to deamidation of Asn-36 to Asp-36.
    • A Ser-36 mutant protein is conformationally stable and resistant to deamidation.
    • This stabilized mutant is advantageous for physical and structural studies of IL-1 alpha.