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

Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
RNA Splicing01:32

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
Alternative RNA Splicing02:18

Alternative RNA Splicing

Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
Exon Recombination02:32

Exon Recombination

The evolution of new genes is critical for speciation. Exon recombination, also known as exon shuffling or domain shuffling, is an important means of new gene formation. It is observed across vertebrates, invertebrates, and in some plants such as potatoes and sunflowers. During exon recombination, exons from the same or different genes recombine and produce new exon-intron combinations, which might evolve into new genes. 
Exon shuffling follows “splice frame rules.” Each exon has three reading...
CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...

You might also read

Related Articles

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

Sort by
Same author

Systematic discovery of UFM1 receptors reveals a regulatory module in DNA repair directing non-homologous end-joining.

Nature communications·2026
Same author

Generating Ultra-Fast Protein trans-Splicing of a Cysteine-Less and Semisynthetic Split Intein for Chemical Protein Labeling.

Chembiochem : a European journal of chemical biology·2026
Same author

Catalysis of Native Chemical Ligation and Expressed Protein Ligation by Alkylselenols.

JACS Au·2025
Same author

Three-Segment Protein Labeling Using a Highly Efficient and Cysteine-Less Split Intein Identified with Computational Prediction of Aggregation Properties.

Angewandte Chemie (International ed. in English)·2025
Same author

Erratum: Optimizing and Evaluating Hand-drawing and Wet-spinning for Recombinant Spider Silk Fiber Production.

Journal of visualized experiments : JoVE·2025
Same author

Optimizing and Evaluating Hand-drawing and Wet-spinning for Recombinant Spider Silk Fiber Production.

Journal of visualized experiments : JoVE·2025
Same journal

Wanted and unwanted modifications of mRNA, and their effect on gene expression and signaling.

The Journal of biological chemistry·2026
Same journal

TGF-β2 drives lipid droplet accumulation in chondrocytes through the TβRI/p-Smad3/Fabp5 axis.

The Journal of biological chemistry·2026
Same journal

Macrophage-specific targeting of histone demethylases with small-molecule inhibitors suppresses inflammatory response in vivo.

The Journal of biological chemistry·2026
Same journal

Substrate and target selectivity of 4'-fluoroadenosine against viral and host polymerases.

The Journal of biological chemistry·2026
Same journal

Correction: Characterization of Mast2 kinase defines structural features, regulation, and substrates.

The Journal of biological chemistry·2026
Same journal

Isotope-Edited ESEEM: A New Method for Probing Copper Binding Sites in Neurodegenerative Proteins.

The Journal of biological chemistry·2026
See all related articles

Related Experiment Video

Updated: May 30, 2026

Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
08:21

Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

Published on: March 16, 2012

Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution.

Julia H Appleby-Tagoe1, Ilka V Thiel, Yi Wang

  • 1Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.

The Journal of Biological Chemistry
|August 12, 2011
PubMed
Summary
This summary is machine-generated.

Directed evolution created novel inteins (protein sequences) that efficiently splice proteins in new locations and hosts. These engineered inteins overcome limitations of natural inteins, enabling broader applications in protein engineering.

More Related Videos

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
09:02

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
09:16

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Related Experiment Videos

Last Updated: May 30, 2026

Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
08:21

Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

Published on: March 16, 2012

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
09:02

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
09:16

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Area of Science:

  • Protein biochemistry
  • Molecular biology
  • Protein engineering

Background:

  • Inteins are protein subsequences that catalyze their own excision and ligation of flanking exteins.
  • Natural and engineered inteins have applications but often show limited activity in non-native environments or leave scars.
  • There is a need for more versatile inteins that function efficiently across diverse protein contexts.

Purpose of the Study:

  • To develop a novel method for engineering inteins with improved and broader functionality.
  • To create inteins that overcome limitations of native inteins, such as reduced activity in non-native hosts and scar formation.

Main Methods:

  • Sequential directed evolution was employed using the Ssp DnaB mini-intein.
  • Mutants were selected for splicing activity at a previously inactive site within a kanamycin resistance protein.
  • The selection and improvement process was iterated for multiple cycles and different insertion sites.

Main Results:

  • Engineered inteins demonstrated high splicing activity at original and novel insertion sites, as well as in different protein contexts.
  • The M86 mutant, with 8 amino acid substitutions, showed a ~60-fold increased protein trans-splicing rate compared to the unevolved split intein.
  • The M86 mutant exhibited an order of magnitude improvement in the binding affinity (K(d)) of its split fragments.

Conclusions:

  • Sequential directed evolution is an effective strategy for generating highly active and versatile inteins.
  • The engineered inteins possess enhanced properties suitable for a wider range of practical applications in protein engineering.
  • The study provides insights into intein structure-function relationships and their evolutionary potential.