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Related Experiment Video

Updated: Jul 10, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Published on: March 16, 2011

Engineering GID4 for use as an N-terminal proline binder via directed evolution.

Svetlana P Ikonomova¹, Bo Yan², Zhiyi Sun²

¹Institute for Bioscience and Biotechnology Research (IBBR), National Institute of Standards and Technology (NIST) and the University of Maryland (UMD), Rockville, Maryland, USA.

Biotechnology and Bioengineering

|October 25, 2024

Summary

Researchers engineered N-terminal amino acid binders (NAABs) for protein sequencing. A modified human protein GID4 shows improved binding to N-terminal proline (Nt-Pro), advancing de novo protein sequencing capabilities.

Keywords:

N‐degron pathway N‐terminal‐amino acid binder (NAAB)directed evolution next‐generation protein sequencing peptide sequencing recognizer

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Published on: February 3, 2022

Area of Science:

Biochemistry
Molecular Biology
Proteomics

Background:

Advancements in nucleic acid sequencing have not been matched in proteomics.
Protein sequencing requires selective N-terminal amino acid binders (NAABs).
Currently, few engineered NAABs meet biotechnology reagent standards.

Purpose of the Study:

To engineer novel NAABs for de novo protein sequencing.
To improve binding affinity and reduce sequence context dependency for N-terminal proline (Nt-Pro) binders.
To establish a viable strategy for engineering NAABs from native binding proteins.

Main Methods:

Engineering of human protein GID4 using directed evolution.
Yeast-surface display and fluorescence-activated cell sorting for variant selection.
Characterization of engineered NAAB variants for Nt-Pro binding.

Main Results:

Identified GID4 variants with enhanced binding response to Nt-Pro.
A specific variant (A252V mutation) demonstrated reduced influence from adjacent amino acid residues.
The engineered NAABs show promise for selective peptide identification.

Conclusions:

The developed workflow is effective for engineering NAABs.
Engineered GID4 variants offer improved Nt-Pro binding for protein sequencing applications.
This approach facilitates the development of essential tools for de novo protein sequencing.