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Related Concept Videos

Protein Modifications in the RER01:26

Protein Modifications in the RER

Modification of secretory and transmembrane proteins entering the rough ER begins in the ER lumen. These modifications aid in protein folding and stabilize the acquired tertiary structure. Protein modifications in the rough ER co-occur at different stages of protein folding.
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Related Experiment Video

Updated: Jul 6, 2026

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture
09:37

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture

Published on: May 2, 2019

Engineering and Adapting Disulfide-Containing Proteins to Enable Intracellular Functionality.

Elise Sylvander1, Michael W Traxlmayr2,3

  • 1Institute of Biochemistry, BOKU University, Vienna, Austria. Elise.sylvander@boku.ac.at.

Methods in Molecular Biology (Clifton, N.J.)
|July 4, 2026
PubMed
Summary

Researchers engineered disulfide-free proteins for intracellular use by replacing cysteine residues. This method enhances protein stability and function in reducing cellular environments, overcoming limitations of natural disulfide bond formation.

Keywords:
Disulfide bond engineeringDisulfide bridgesProtein engineeringProtein stabilityYeast surface display

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High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
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High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Published on: July 30, 2014

Area of Science:

  • Biochemistry
  • Protein Engineering
  • Molecular Biology

Background:

  • Disulfide bonds are crucial for protein structure and stability but do not form in the reducing intracellular environment.
  • Intracellular proteins lacking disulfide bonds often exhibit misfolding, instability, and reduced function.
  • Engineering disulfide-free variants is necessary to restore stability and function for intracellular applications.

Purpose of the Study:

  • To develop a method for creating stable and functional disulfide-free protein variants.
  • To enable intracellular applications of proteins that normally require disulfide bonds.
  • To provide a screening strategy for identifying improved disulfide-free variants.

Main Methods:

  • Utilized site-directed mutagenesis to substitute cysteine residues with optimal amino acid combinations.
  • Employed yeast surface display for high-throughput screening of protein variants.
  • Developed protocols for screening thermal stability using conformationally specific ligands.
  • Explored correlation between protein stability and expression levels in yeast as an alternative screening metric.

Main Results:

  • Successfully engineered disulfide-free protein variants with enhanced stability and function.
  • Demonstrated the efficacy of yeast surface display for screening protein stability.
  • Provided detailed protocols for direct thermal stability screening and indirect screening via expression levels.
  • Discussed strategies for randomizing cysteine residues and managing proteins with multiple disulfide bonds.

Conclusions:

  • The presented method enables the engineering of stable and functional disulfide-free proteins for intracellular use.
  • Yeast surface display coupled with thermal stability screening is an effective approach for protein engineering.
  • The developed protocols offer versatile strategies for improving protein characteristics in reducing environments.