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Updated: Dec 3, 2025

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Published on: July 3, 2016

High-Throughput Protein Engineering by Massively Parallel Combinatorial Mutagenesis.

Yuk Kei Wan¹, Gigi C G Choi¹, Alan S L Wong^2,3

¹Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong SAR, China.

Methods in Molecular Biology (Clifton, N.J.)

|October 30, 2020

Summary

This summary is machine-generated.

This study introduces CombiSEAL, a high-throughput platform for protein engineering. It efficiently generates and screens combinatorial mutants to optimize protein functions and analyze mutation effects.

Keywords:

CombiSEAL Combinatorial genetics en masse Combinatorial mutagenesis High-throughput screening Next-generation sequencing Protein engineering Protein variant characterization

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Last Updated: Dec 3, 2025

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Area of Science:

Biochemistry and Molecular Biology
Synthetic Biology
Protein Engineering

Background:

Optimizing protein function through combinatorial mutations is crucial for protein engineering.
The vastness of the combinatorial space makes identifying optimal variants challenging without efficient screening methods.

Purpose of the Study:

To develop a high-throughput gene assembly and screening strategy for combinatorial protein engineering.
To introduce the CombiSEAL platform for modular mutagenesis, barcoding, and seamless ligation of protein segments.

Main Methods:

Modularization of protein sequences into segments for mutagenesis and barcoding.
Seamless single-pot ligation of different segments to create combination mutant libraries.
Next-generation sequencing for high-throughput tracking of protein variant activities.

Main Results:

CombiSEAL enables the generation of large libraries of combination mutants.
High-throughput tracking of variant activities during selection processes is achieved via barcode sequencing.
The platform facilitates the identification of improved protein variants.

Conclusions:

CombiSEAL is an effective strategy for combinatorial protein engineering.
It allows for systematic analysis of beneficial, deleterious, and neutral effects of combined mutations.
This platform aids in guiding protein engineering efforts by optimizing protein functions.