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Updated: Jun 4, 2026

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
Focusing mutations within random libraries to distinct areas: protein domain library generation by overlap extension
1Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, Düsseldorf, Germany.
Abstract:
Directed evolution is an often used approach toward new proteins with tailor-made properties. It consists of random variation of the coding sequence of a protein followed by an appropriate selection procedure or a suitable type of property read out. In many, if not all cases, it is of significant advantage to constrain the randomly mutagenized DNA sequence to that encoding a particular part of the protein or a distinct domain, and not to mutate the entire gene of the target protein. For this purpose, a three-step, polymerase-based method was developed, which is independent of two flanking restriction sites adjacent to the nucleotide sequence supposed to be mutagenized, and named protein library generation by overlap extension (PDLGO).
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