Creating Randomized Amino Acid Libraries with the QuikChange
            <sup>®</sup>
            Multi Site-Directed Mutagenesis Kit

Hogrefe, Holly H.; Cline, Janice; Youngblood, Geri L.; Allen, Ronda M.

doi:10.2144/02335pf01

Cited by 164 publications

(157 citation statements)

References 18 publications

(24 reference statements)

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“…The library generated by saturation mutagenesis at positions 93∕94 by using NDT codon degeneracy was found to contain two mutants that catalyze measurable conversion of ketone rac-1d within a reaction time of 24 h as specified in the GC screening process, namely Gln93Val/ Pro94Phe and Gln93Asn/Pro94Asp. Due to amino acid bias inherent in the nature of the saturation mutagenesis protocol (14,31), the possibility that hits were missed cannot be excluded inspite of the high percentage of coverage. Because the double mutant Gln93Asn/Pro94Asp proved to be the much more active of the two identified hits, we subsequently focused all further efforts on this variant.…”

Section: Resultsmentioning

confidence: 99%

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Acevedo

Reetz

2010

Proc. Natl. Acad. Sci. U.S.A.

121

118

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The molecular basis of allosteric effects, known to be caused by an effector docking to an enzyme at a site distal from the binding pocket, has been studied recently by applying directed evolution. Here, we utilize laboratory evolution in a different way, namely to induce allostery by introducing appropriate distal mutations that cause domain movements with concomitant reshaping of the binding pocket in the absence of an effector. To test this concept, the thermostable Baeyer-Villiger monooxygenase, phenylacetone monooxygenase (PAMO), was chosen as the enzyme to be employed in asymmetric Baeyer-Villiger reactions of substrates that are not accepted by the wild type. By using the known X-ray structure of PAMO, a decision was made regarding an appropriate site at which saturation mutagenesis is most likely to generate mutants capable of inducing allostery without any effector compound being present. After screening only 400 transformants, a double mutant was discovered that catalyzes the asymmetric oxidative kinetic resolution of a set of structurally different 2-substituted cyclohexanone derivatives as well as the desymmetrization of three different 4-substituted cyclohexanones, all with high enantioselectivity. Molecular dynamics (MD) simulations and covariance maps unveiled the origin of increased substrate scope as being due to allostery. Large domain movements occur that expose and reshape the binding pocket. This type of focused library production, aimed at inducing significant allosteric effects, is a viable alternative to traditional approaches to "designed" directed evolution that address the binding site directly.allosteric effects | enzymes | molecular dynamics simulations | protein engineering P rotein allostery has been recognized as a positive or negative cooperative event, leading to a structural change at the binding site as a result of distal docking of a molecule acting as an effector (1-5). Allosteric effects can be influenced by such factors as variation in pH, temperature, ionic strength, and covalent modification as well as mutational changes. A variety of experimental and computational techniques have been utilized to unravel the intricacies of this phenomenon (1-5), but also to achieve useful applications such as the creation of protein switches (6). Among the techniques that have been applied to address these challenges is directed evolution, a method that is generally used to engineer the catalytic profiles of enzymes or the binding properties of proteins (7-11). In the endeavor to make directed evolution of enantioselective enzymes more efficient than in the past, we recently introduced the concept of iterative saturation mutagenesis according to which sites around the binding pocket are randomized iteratively, a given site being composed of one or more amino acid positions in the protein (12-14.) When applying this technique to enzymes displaying strong allosteric effects or coupled motions along the reaction coordinate (15), it is necessary to consider such phenomena to make reasonable...

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Section: Resultsmentioning

confidence: 99%

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Acevedo

Reetz

2010

Proc. Natl. Acad. Sci. U.S.A.

121

118

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“…In terms of saturation mutagenesis, the pair of primers need not contain a single mutation, but may instead contain a degenerate codon or indeed take the form of a DNA cassette as described in section 3.1.1. To expand the technology further, the QuikChange® Multi Site-Directed Mutagenesis Kit has been developed to target up to five sites simultaneously (Hogrefe, Cline, Youngblood & Allen, 2002). Target residues have to be at least 5 codons apart and so automatically exclude targeting contiguous sites.…”

Section: Simple Primer-extension Mutagenesismentioning

confidence: 99%

Beyond the Natural Proteome

Amaral

Frigotto²,

Hine

2017

Methods in Enzymology

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“…Oligonucleotide-directed SDM methods have been described, including PCR-based methods, and Adachi and Fukuhara, 2012 methods based on plasmid DNA templates and mutant DNA (Hogrefe et al, 2002). Most of these methods are used to introduce only one mutation site at a time.…”

Section: Multi-sdm By Multiple Mutagenic Oligonucleotide-directed (Mmmentioning

confidence: 99%

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

Zhang

2016

J. Zhejiang Univ. Sci. B

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Abstract:With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

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Creating Randomized Amino Acid Libraries with the QuikChange ^® Multi Site-Directed Mutagenesis Kit

Cited by 164 publications

References 18 publications

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Beyond the Natural Proteome

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

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Creating Randomized Amino Acid Libraries with the QuikChange ® Multi Site-Directed Mutagenesis Kit

Cited by 164 publications

References 18 publications

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Beyond the Natural Proteome

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

Contact Info

Product

Resources

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Creating Randomized Amino Acid Libraries with the QuikChange ^® Multi Site-Directed Mutagenesis Kit