Mutation Maker, An Open Source Oligo Design Platform for Protein Engineering

Abstract

Protein engineering is the discipline of developing useful proteins for applications in research, therapeutic, and industrial processes by modification of naturally occurring proteins or by invention of de novo proteins. Modern protein engineering relies on the ability to rapidly generate and screen diverse libraries of mutant proteins. However, design of mutant libraries is typically hampered by scale and complexity, necessitating development of advanced automation and optimization tools that can improve efficiency and accuracy. At present, automated library design tools are functionally limited or not freely available. To address these issues, we developed Mutation Maker, an open source mutagenic oligo design software for large-scale protein engineering experiments. Mutation Maker is not only specifically tailored to multisite random and directed mutagenesis protocols, but also pioneers bespoke mutagenic oligo design for de novo gene synthesis workflows. Enabled by a novel bundle of orchestrated heuristics, optimization, constraint-satisfaction and backtracking algorithms, Mutation Maker offers a versatile toolbox for gene diversification design at industrial scale. Supported by in silico simulations and compelling experimental validation data, Mutation Maker oligos produce diverse gene libraries at high success rates irrespective of genes or vectors used. Finally, Mutation Maker was created as an extensible platform on the notion that directed evolution techniques will continue to evolve and revolutionize current and future-oriented applications.

Development of antiviral carbon quantum dots that target the Japanese encephalitis virus envelope protein

“…The constructed pET30a (+) plasmids containing the E segment of JEV (designated as pET30a(+)-E). The pET30a(+)-E-Mut-S123R, pET30a(+)-E-Mut-K312R, and pET30a(+)-E-Mut-S123R/K312R were produced using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) by following the manufacturer's protocol ( 89 ).…”

“…The pET-JEV E, pET-E-Mut-S123R, pET-E-Mut-K312R, and pET-E-Mut-S123R/K312R plasmids respectively transformed into E. coli BL21 (Medical Supply Company Ltd) and induced target protein expression by 0.6 mM isopropyl β-D-1-thiogalactopyranoside in Luria-Bertani (LB) medium containing kanamycin (final concentration: 50 μg ml −1 ) at 25 °C for 16 h. His-JEV E recombinant protein and the other proteins with mutation sites were purified by column chromatography on Ni sepharose 6 fast flow (GE Healthcare). The induction and purification of recombinant protein refer to the manufacturer's protocol and previous studies ( 89 ).…”

Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan

“…The authors have cited additional references within the Supporting Information. [32][33][34][35][36][37][38][39]…”

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

“…Mutation Maker [138] and GeneGenie [139] serve as oligo design platforms for protein engineering. Taking Mutation Maker as an example, the platform designs oligos for various applications, including site-scanning saturation mutagenesis, multi-site-directed mutagenesis, and PCR-based accurate gene synthesis.…”