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Expression cloning of cDNA by phage display selection

J Light¹, R Maki, N Assa-Munt

¹The Burnham Institute, La Jolla, CA 92037, USA. light@ljcrf.edu

Nucleic Acids Research

|November 1, 1996

Summary

Phage display technology enabled the successful expression cloning of a mouse kappa chain fragment. This method efficiently identifies specific protein fragments from large cDNA libraries for research applications.

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Area of Science:

Molecular Biology
Immunology
Biotechnology

Background:

Phage display is a powerful technique for isolating specific protein interactions.
Expression cloning allows for the identification and recovery of cDNA fragments encoding desired proteins.
Mouse kappa chain fragments are important components of antibodies.

Purpose of the Study:

To demonstrate the utility of phage display for expression cloning of mouse kappa chain fragments.
To establish a method for selecting specific cDNA inserts from a library using protein binding.

Main Methods:

Constructed a cDNA library from mouse.
Utilized filamentous phage display to express proteins anchored by a synthetic leucine zipper.
Performed affinity selection using anti-mouse Fab antibodies to isolate target clones.
Screened a library of 4 x 10^6 transformants over four selection rounds.

Main Results:

Successfully expression cloned a mouse kappa chain fragment.
Identified two distinct clones with different cDNA insert sizes after selection.
Confirmed the efficacy of the leucine zipper for anchoring proteins to phage.

Conclusions:

Phage display is a highly effective method for cDNA expression cloning.
This technique can be applied to isolate specific antibody fragments.
The developed method offers a robust approach for protein discovery and characterization.

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