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Engineering stable cytoplasmic intrabodies with designed specificity.

Marcello Donini1, Veronica Morea, Angiola Desiderio

  • 1ENEA, UTS Biotecnologie, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, P.O. Box 2400 I-00100, Roma, Italy.

Journal of Molecular Biology
|June 26, 2003
PubMed
Summary
This summary is machine-generated.

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Researchers engineered stable antibody fragments, called intrabodies, for cytoplasmic expression. Grafting antigen-binding regions onto a stable scaffold successfully transferred specificity, enabling custom intrabody development.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Immunology

Background:

  • Developing stable antibody fragments for cytoplasmic expression (intrabodies) remains challenging.
  • The scFv(F8) antibody fragment exhibits high stability and functional folding in prokaryotic and eukaryotic cytoplasm.

Purpose of the Study:

  • To investigate the contribution of different regions to the cytoplasmic stability and specificity of scFv(F8).
  • To create chimeric antibody fragments by grafting antigen-binding residues from a less stable fragment (scFv(D1.3)) onto the stable scFv(F8) scaffold.

Main Methods:

  • Construction of five chimeric scFv molecules (scFv-P1 to P5).
  • Expression of chimeric scFvs in the periplasm and cytoplasm of Escherichia coli.
  • Analysis of hen egg lysozyme (HEL) binding affinity of periplasmic and cytoplasmic forms.

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Main Results:

  • All five chimeric scFvs were expressed solubly in both periplasm and cytoplasm.
  • Periplasmic oxidized forms and cytoplasmic scFv(P3) (reducing conditions) showed high HEL binding affinity (K(d) ≈ 15-16 nM).
  • Antigen-binding properties of scFv(D1.3) were successfully transferred to the scFv(F8) scaffold.

Conclusions:

  • The scFv(F8) scaffold can accommodate and functionally display grafted antigen-binding regions.
  • This approach facilitates the engineering of novel intrabodies with specificities tailored for cytoplasmic targeting.