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Intrabody construction and expression III: engineering hyperstable V(H) domains.

P Wirtz1, B Steipe

  • 1Genzentrum der Ludwig-Maximilians-Universität, München, Germany.

Protein Science : a Publication of the Protein Society
|December 14, 1999
PubMed
Summary
This summary is machine-generated.

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Stability engineering enables functional expression of immunoglobulin V(H) domains in the reducing cytoplasm. Rational design using consensus sequences improved V(H) domain stability and solubility for potential intrabody development.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Immunoglobulin domains require disulfide bonds for proper folding, limiting their expression in the reducing cellular cytoplasm.
  • Previous work demonstrated successful cytoplasmic expression of immunoglobulin V(L) domains through stability engineering.

Purpose of the Study:

  • To apply rational stability engineering via consensus sequence analysis to immunoglobulin V(H) domains.
  • To overcome challenges associated with V(H) domain aggregation, refolding, and in vitro handling.

Main Methods:

  • Rational stability engineering using consensus sequence analysis was applied to V(H) domains.
  • Point mutations were predicted and experimentally verified to enhance V(H) domain stability.
  • The stabilized V(H) domain was expressed in Escherichia coli cytoplasm and reconstituted with a V(L) domain.

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Main Results:

  • Several stabilizing point mutations were identified and validated in a designed catalytic Fv fragment's V(H) domain.
  • The mutations were additive, leading to a prototype domain with enhanced stability against denaturation and increased thermal half-life.
  • The stabilized V(H) domain was expressed solubly in E. coli cytoplasm at ~1.2 mg/L and remained functional upon reconstitution.

Conclusions:

  • Consensus sequence engineering is a rational approach for creating stable, soluble V(H) domains.
  • This method facilitates the cytoplasmic expression of functional V(H) domains, previously a significant challenge.
  • The findings support the development of intrabodies through engineered immunoglobulin domains.