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 Concept Videos

Antibiotic Selection00:57

Antibiotic Selection

53.4K
Overview
53.4K
Molecular Chaperones and Protein Folding03:00

Molecular Chaperones and Protein Folding

17.9K
The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
The...
17.9K

You might also read

Related Articles

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

Sort by
Same author

Biology of host-dependent restriction-modification in prokaryotes.

EcoSal Plus·2025
Same author

A galactose-based auto-expression system improves T7-inducible protein production in Escherichia coli.

Scientific reports·2025
Same author

Plasmid Library Construction From Genomic DNA.

Current protocols·2025
Same author

Domainator, a flexible software suite for domain-based annotation and neighborhood analysis, identifies proteins involved in antiviral systems.

Nucleic acids research·2024
Same author

Revisiting the y-ome of Escherichia coli.

Nucleic acids research·2024
Same author

MicroRNA-148a Targets <i>DNMT1</i> and <i>PPARGC1A</i> to Regulate the Viability, Proliferation, and Milk Fat Synthesis of Ovine Mammary Epithelial Cells.

International journal of molecular sciences·2024

Related Experiment Video

Updated: Jun 30, 2025

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions
07:03

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Published on: December 23, 2022

2.9K

SAS: Split antibiotic selection for identifying chaperones that improve protein solubility.

Emily McNutt1, Na Ke1, Alexandre Thurman1

  • 1New England Biolabs, 240 County Road, Ipswich, MA 01938, USA.

Heliyon
|March 18, 2024
PubMed
Summary

Researchers developed a Split Antibiotic Selection (SAS) system to improve recombinant protein solubility in E. coli. This method links protein folding to cell viability, enabling efficient selection of beneficial chaperones for enhanced protein production.

Keywords:
ChaperonesE. coliGenetic selectionRecombinant protein expression

More Related Videos

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo
08:32

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Published on: October 23, 2016

10.6K
Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay
06:51

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Published on: July 21, 2021

2.8K

Related Experiment Videos

Last Updated: Jun 30, 2025

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions
07:03

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Published on: December 23, 2022

2.9K
Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo
08:32

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Published on: October 23, 2016

10.6K
Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay
06:51

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Published on: July 21, 2021

2.8K

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Protein Engineering

Background:

  • Heterologous protein expression in Escherichia coli is crucial for research and industry.
  • Achieving soluble and active recombinant proteins in E. coli presents significant challenges.
  • Current methods for enhancing protein solubility are often time-consuming and lack a universal solution.

Purpose of the Study:

  • To develop a novel genetic system for selecting protein folding factors.
  • To link protein solubility directly to cellular viability for efficient screening.
  • To create a versatile tool for improving recombinant protein production.

Main Methods:

  • Developed a Split Antibiotic Selection (SAS) system using a tripartite fusion with aminoglycoside 7″-phosphotransferase-Ia (APH(7″)).
  • Inserted target proteins in-frame within the APH(7″) coding sequence to create a solubility-dependent hygromycin B resistance.
  • Utilized a chaperone library for pooled genetic selection to identify solubility-enhancing factors.

Main Results:

  • Demonstrated SAS functionality with human mitochondrial Hsp70 ATPase domain and its co-chaperone Hep.
  • Showcased that cellular hygromycin B resistance directly correlates with the solubility of the tripartite fusion protein.
  • Successfully identified chaperones that improve client protein solubility using pooled SAS screening.

Conclusions:

  • The tripartite APH(7″) fusion system effectively links in vivo protein solubility to hygromycin B resistance.
  • SAS enables selection of chaperones that enhance the solubility of various client proteins.
  • This system streamlines the identification of optimal folding conditions, reducing the need for individual testing.