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A native-like artificial protein from antisense DNA.

Nicolas Fischer1, Lutz Riechmann, Greg Winter

  • 1Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. nfischer@novimmune.com

Protein Engineering, Design & Selection : PEDS
|February 27, 2004
PubMed
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Researchers created stable, folded chimeric proteins using antibody fragments and random human DNA. One protein, 2a6, is protease-resistant and exhibits native-like folding, suggesting antisense DNA can evolve new proteins.

Area of Science:

  • Protein engineering
  • Molecular biology
  • Biochemistry

Background:

  • Antibody variable domains contain beta-sheets that can be used as protein scaffolds.
  • Random polypeptide segments from human cDNA can be combined with designed segments.
  • Display technologies allow for the selection of functional proteins from large libraries.

Purpose of the Study:

  • To create novel folded chimeric proteins by combining antibody-derived beta-sheet segments with random human cDNA fragments.
  • To select and characterize these chimeric proteins for stability and folding properties.
  • To investigate the potential of antisense DNA sequences as a source for protein evolution.

Main Methods:

  • Construction of a phage display library combining a designed beta-sheet polypeptide (bait) with random human cDNA fragments.

Related Experiment Videos

  • Selection of folded polypeptides using proteolysis.
  • Expression, purification, and characterization of selected chimeric proteins (e.g., 2a6) using techniques like heat treatment, circular dichroism (CD) spectroscopy, and differential scanning calorimetry (DSC).
  • Sequence analysis to identify the origin of the cDNA segments.

    • Main Results:

    • Successful creation and selection of folded chimeric proteins, exemplified by protein 2a6.
    • Protein 2a6 was expressed in E. coli cytoplasm, purified by heat treatment (90°C), and found to be soluble, dimeric, and protease-resistant.
    • CD spectrum indicated a mix of alpha and beta structures.
    • 2a6 exhibited cooperative and reversible unfolding with a folding energy of 11.4 kcal mol(-1), characteristic of native proteins.
    • Refolding occurred without aggregation.

    Conclusions:

    • Chimeric proteins can be engineered using antibody beta-sheet scaffolds and random cDNA fragments.
    • Antisense DNA sequences can serve as a source for the evolution of soluble, stable, and functional proteins.
    • The selected protein 2a6 demonstrates native-like stability and folding properties, validating the approach.