Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

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

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.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Advances in Cell Signaling Pathways: A Comprehensive Review

Journal of Cellular Biology·2024
Same author

Novel Approaches to Tissue Engineering and Regenerative Medicine

Nature Methods·2023
Same author

Understanding Molecular Mechanisms in Disease Progression

Cell Reports·2023
Same author

Genomic Profiling Reveals New Biomarkers for Early Diagnosis

Nature Genetics·2023
Same author

CRISPR-Based Screening Identifies Key Regulators of Cell Growth

Cell Reports·2022
Same author

Structural Insights into Membrane Protein Function

Journal of Cellular Biology·2022

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.

Related Experiment Videos

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.