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Updated: Dec 14, 2025

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Published on: October 3, 2018

An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation.

Vaishali Verma¹, Gopal Joshi¹, Amita Gupta^1,2

¹Centre for Innovation in Infectious Disease Research, Education and Training (CIIDRET), University of Delhi South Campus, New Delhi, India.

|July 24, 2020

Summary

This summary is machine-generated.

This study introduces a novel system for selecting correct open-reading frames (ORFs) in DNA fragment libraries, significantly improving protein expression efficiency. The method utilizes split beta-lactamase complementation for highly accurate ORF selection and seamless cloning.

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Last Updated: Dec 14, 2025

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

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Published on: October 3, 2018

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Area of Science:

Molecular Biology
Protein Expression
Biotechnology

Background:

Genome-scale protein expression faces challenges like poor solubility and large protein sizes.
Random DNA fragmentation for protein fragment libraries yields low efficiency due to incorrect open-reading frames (ORFs).

Purpose of the Study:

To develop a highly efficient system for selecting correct ORFs in DNA fragment libraries.
To enable seamless transfer of selected DNA fragments into downstream vector systems.
To overcome limitations in genome-scale protein expression and purification.

Main Methods:

Utilized split beta-lactamase protein fragment complementation for ORF selection.
Implemented a restriction enzyme-free cloning strategy for seamless DNA fragment transfer.
Applied the system to model constructs and a 30-gene DNA fragment library from M. tuberculosis.

Main Results:

Achieved near 100% selection accuracy for correct ORFs in model constructs.
Successfully constructed and validated an ORF-selected DNA fragment library for M. tuberculosis.
Demonstrated successful cytosolic expression of ORF-selected protein fragments in E. coli.

Conclusions:

The developed ORF selection system significantly enhances the efficiency of generating functional protein fragments.
The restriction enzyme-free cloning strategy facilitates versatile application in various downstream vector systems.
This approach provides a robust solution for challenges in large-scale protein expression and functional studies.