A statistical analysis of random mutagenesis methods used for directed protein evolution.

@article{Wong2006ASA,
  title={A statistical analysis of random mutagenesis methods used for directed protein evolution.},
  author={Tuck Seng Wong and Danilo Roccatano and Martin Zacharias and Ulrich Schwaneberg},
  journal={Journal of molecular biology},
  year={2006},
  volume={355 4},
  pages={
          858-71
        }
}
View on PubMed
classes.soe.ucsc.edu
147 Citations
Highly Influential Citations
4
Background Citations
34
Methods Citations
21
Results Citations
2

Figures and Tables from this paper

147 Citations

The Mutagenesis Assistant Program.
TLDR
The latest development of the program, the MAP(2.0)3D server, correlates the generated amino acid substitution patterns of a specific random mutagenesis method to the sequence and structural information of the target protein, helping to select an experimental strategy that improves the chances of obtaining functionally efficient and/or stable enzyme variants.
MAP(2.0)3D: a sequence/structure based server for protein engineering.
TLDR
A completely renewed and improved version of the Mutagenesis Assistant Program server, the MAP(2.0)3D server, which correlates the generated amino acid substitution patterns to the structural information of the target protein, which aids in the selection of a more suitable random mutagenesis method with specific biases on amino acids substitution patterns.
Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM‐3 heme domain
TLDR
A detailed analysis for the P450 BM‐3 heme domain showed that an ideal transversion bias generates more diverse amino acid substitution patterns with a significantly different chemical composition than an ideal transition bias.
Challenges of the genetic code for exploring sequence space in directed protein evolution
TLDR
The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments.
Enzyme Engineering by Directed Evolution
TLDR
This chapter treats exclusively directed evolution of proteins, the primary focus being on recent methodology developments and the reasons for the apparent efficacy of iterative combinatorial active-site saturation testing as a form of iteratives saturation mutagenesis (ISM).
Directed Evolution of DNA Polymerases for Advancement of the SeSaM Mutagenesis Method and Biotransformations with P450 BM3 Monooxygenase
TLDR
The protein engineering work comprised development of a novel high-throughput screening system for non-processive DNA polymerases, screening and identification of Dpo4 variants capable of directed evolution, and selection of one candidate (Dpo4 from Sulfolobus solfataricus) for directed evolution.
Directed Evolution by Using Iterative Saturation Mutagenesis Based on Multiresidue Sites
TLDR
In this study, ISM is applied for the first time by using randomization sites composed of five amino acid positions, and notable activity and 99 % diastereoselectivity were obtained, indicating that such an ISM strategy is viable in protein engineering.
A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons.
  • J. Liu, T. Cropp
  • Biology
    Protein engineering, design & selection : PEDS
  • 2012
TLDR
Key to this method is the development of a Mu transposon variant with asymmetric terminal sequences, which allows for mutations of one, two or three consecutive codons.
Advancements of the Sequence Saturation Mutagenesis (SeSaM) Method to Efficiently Explore Protein Sequence Space
TLDR
Sequence Saturation Mutagenesis (SeSaM-Tv+) is a novel random mutagenesis method which can construct transversion biased mutant library and consecutive mutations and was reported to achieve enrichment of desired transversions.
Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations
TLDR
The diversity in random mutagenesis libraries employed in directed evolution experiments was determined and the chemical composition of the amino acid substitutions differed, however, significantly from the two epPCR methods to SeSaM‐Tv P/P.
...
...

References

SHOWING 1-10 OF 46 REFERENCES
The diversity challenge in directed protein evolution.
TLDR
This review summarizes, categorizes and discusses advantages and disadvantages of random mutagenesis methods used for generating genetic diversity, and provides a guide for developing successful directed evolution strategies by selecting complementary methods for generating diverse mutant libraries.
How enzymes adapt: lessons from directed evolution.
  • F. Arnold
  • Biology
    Trends in biochemical sciences
  • 2001
A new approach to random mutagenesis in vitro
TLDR
This is the first application of EMS for in vitro mutagenesis of a defined DNA sequence and among the point mutations detected, GC → AT transition accounts for 42.1%, AT → GC transition 34.2% and GC/CG transversion 23.7%, respectively.
Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.
A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro.
A new efficient in vitro mutagenesis method for the generation of complete random mutant libraries, containing all possible single base substitution mutations in a cloned gene is described. The…
Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization.
TLDR
A PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency is described and its natural logarithm is proportional to the average mutation frequency of the populations.
Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I
TLDR
It is demonstrated that the error-prone Pol I can be used to generate enzymes with distinct properties by generating TEM-1 β-lactamase mutants able to hydrolyze a third-generation lactam antibiotic, aztreonam.
Low-fidelity Pyrococcus furiosus DNA polymerase mutants useful in error-prone PCR.
TLDR
Mutants of the family B DNA polymerase from Pyrococcus furiosus (Pfu-Pol), with superb performance in error-prone PCR, are described, which are anticipated to be extremely useful for the randomization of gene, and hence protein, sequences.
Sensitive Assay for Laboratory Evolution of Hydroxylases toward Aromatic and Heterocyclic Compounds
TLDR
A medium-throughput assay system designed for screening mutant libraries of oxygenase capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases is reported.
Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions.
Very complex mutant libraries of the dihydrofolate reductase (DHFR) gene encoded by the Escherichia coli plasmid R67 were created using hypermutagenic PCR with biased deoxynucleotide triphosphate…
...
...