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Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing
Published on: October 3, 2018
Protein domain mapping by lambda phage display: the minimal lactose-binding domain of galectin-3
T Moriki1, I Kuwabara, F T Liu
1Department of Cell Biology, Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California, 92037, USA.
Biochemical and Biophysical Research Communications
|November 24, 1999
Summary
Researchers identified the minimal carbohydrate-binding domain of human galectin-3 (Gal-3) using bacteriophage lambda surface display. This method efficiently maps protein domains and studies molecular interactions without purified proteins.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Understanding protein structure-function relationships requires mapping functional domains.
- Human galectin-3 (Gal-3) is a protein with known carbohydrate-binding properties.
Purpose of the Study:
- To determine the minimal carbohydrate-binding domain of human galectin-3 (Gal-3).
- To establish bacteriophage lambda surface display as a tool for protein domain mapping and interaction studies.
Main Methods:
- Generated a phage display library of human Gal-3 cDNA using random DNase I digestion and cloning into the lambdafoo vector.
- Screened the library by affinity selection using lactose immobilized on agarose beads.
- Analyzed DNA sequences of isolated clones to identify the minimal binding domain and determined dissociation constants in solution.
Main Results:
- Identified a 136-amino-acid minimal folding domain of Gal-3 responsible for lactose binding.
- Quantified relative dissociation constants for lactose binding to the Gal-3 minimal domain displayed on phage.
- Demonstrated the utility of bacteriophage lambda surface display for mapping protein domains.
Conclusions:
- The minimal lactose-binding domain of human galectin-3 comprises 136 amino acids.
- Bacteriophage lambda surface display is an effective technique for protein domain mapping.
- This method facilitates in vitro studies of macromolecular interactions without requiring purified or labeled proteins.
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