Related Experiment Video
Updated: Jun 28, 2026
04:18
A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction
Published on: May 17, 2024
The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning
Nick S Berrow1, David Alderton, Raymond J Owens
1Oxford Protein Production Facility, Welcome Trust Centre for Human Genetics, Oxford, UK.
Methods in Molecular Biology (Clifton, N.J.)
|November 7, 2008
Summary
This chapter details In-Fusion PCR cloning for seamless DNA joining, enabling the creation of versatile expression vectors. These vectors facilitate protein expression with engineered His-tags across multiple hosts.
Area of Science:
- Molecular Biology
- Recombinant DNA Technology
Background:
- Efficient gene cloning is crucial for protein expression studies.
- Traditional ligation-based cloning can be time-consuming and inefficient.
Purpose of the Study:
- To present protocols for constructing expression vectors using In-Fusion PCR cloning.
- To describe the design of multi-host compatible vectors for protein expression.
- To detail the engineering of His-tags for precise protein purification.
Main Methods:
- In-Fusion PCR cloning for ligation-independent joining of vector and insert DNA.
- Vector preparation and PCR amplification of target sequences.
- Detailed procedure for inserting PCR products into vectors using In-Fusion.
Main Results:
- Seamless joining of DNA sequences without traditional ligation.
- Development of a suite of expression vectors compatible with multiple host systems.
- Successful engineering of His-tags for controlled protein expression and purification.
Conclusions:
- In-Fusion PCR cloning offers an efficient method for expression vector construction.
- The developed vectors provide a versatile platform for recombinant protein expression.
- Precise His-tag engineering enhances protein purification strategies.
