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Target-assisted iterative screening of phage surface display cDNA libraries.
Alexei Kurakin1, Susan Wu, Dale E Bredesen
1Buck Institute for Age Research, Novato, CA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|March 17, 2004
Summary
Target-assisted iterative screening (TAIS) is a rapid, two-step method for identifying specific molecular binders. This protocol details its use with a T7 phage-displayed complementary DNA (cDNA) library to discover protein-protein interactions.
Area of Science:
- Molecular Biology
- Biochemistry
- Protein Interactions
Background:
- Identifying specific molecular binders is crucial for understanding biological processes.
- Existing affinity selection methods can be time-consuming and complex.
- Novel methods are needed for efficient screening of large molecular libraries.
Purpose of the Study:
- To describe and validate the target-assisted iterative screening (TAIS) protocol.
- To demonstrate the application of TAIS for identifying protein-protein interactions.
- To provide a detailed method for researchers interested in molecular binder discovery.
Main Methods:
- Target-assisted iterative screening (TAIS) involves a two-step in vitro affinity selection process.
- Application of TAIS to a T7 phage-displayed complementary DNA (cDNA) library.
- Purification of the target protein as a glutathione-S-transferase (GST) fusion protein.
- Modification of the purified target protein for optimal screening.
Main Results:
- TAIS enables rapid and efficient selection of specific binders from vast molecular diversities.
- The protocol successfully applied TAIS to identify binders for a protein domain target.
- Detailed procedures for target purification and modification were established.
Conclusions:
- TAIS is a powerful and versatile method for in vitro affinity selection.
- This protocol facilitates the identification and characterization of novel protein-protein interactions.
- TAIS is a method of choice for researchers studying peptide recognition domains.