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Related Experiment Video

Updated: Jun 8, 2026

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

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Published on: January 8, 2015

Self-cleaving purification tags re-engineered for rapid Topo® cloning.

Wan-Yi Wu¹, Alison R Gillies, Judy F Hsii

¹Dept. of Chemical Engineering, Princeton University, Engineering Quadrangle, Princeton, NJ 08540, USA.

Biotechnology Progress

|October 15, 2010

Summary

Engineered inteins now seamlessly integrate with Topo® cloning systems, enhancing protein expression vector construction. These modified inteins maintain self-cleaving functions for versatile affinity tag applications.

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Area of Science:

Molecular Biology
Protein Engineering
Biotechnology

Background:

Inteins are protein splicing elements crucial for protein engineering.
Previous intein variants (ΔI-CM, ΔI(G)-CM) required modification for broader cloning compatibility.
Invitrogen's Topo® cloning offers a rapid, one-step method for vector construction.

Purpose of the Study:

To re-engineer existing inteins for compatibility with the Topo® cloning system.
To maintain the self-cleaving and conditional characteristics of the inteins.
To enhance the versatility of intein-mediated protein expression strategies.

Main Methods:

Rational re-engineering of ΔI-CM and ΔI(G)-CM inteins.
Addition of the vaccinia virus topoisomerase I DNA recognition sequence (TCCTT) to the 3' end of inteins.
Characterization of the modified inteins' self-cleaving activity, pH, and temperature sensitivity.
Assessment of compatibility with Topo® and Gateway® cloning systems.

Main Results:

Successfully created Topo®-compatible inteins with a TCCTT recognition sequence.
Modified inteins exhibited an altered C-terminal amino acid sequence (VVVHN to VLVHN).
The engineered inteins retained self-cleaving function with pH and temperature sensitivity.
Cleavage rate was slightly reduced but completed within hours under optimal conditions (pH 6.5, 37°C).
The modified ΔI(GT)-CM intein demonstrated simultaneous compatibility with both Topo® and Gateway® cloning methods.

Conclusions:

Engineered inteins expand the utility of intein-mediated technology for protein expression.
The new inteins facilitate rapid, parallel construction of diverse expression vectors.
This advancement enables efficient exploration of combinations between target genes, affinity tags, and expression hosts.