Advances in generating functional diversity for directed protein evolution

Shivange, Amol V.; Marienhagen, Jan; Mundhada, Hemanshu; Schenk, Alexander; Schwaneberg, Ulrich

doi:10.1016/j.cbpa.2009.01.019

Cited by 154 publications

(102 citation statements)

References 39 publications

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“…[1][2][3] For instance, intermolecular interactions can lead to significant aggregation and subsequent precipitation, a phenomenon which is difficult to predict. Directed evolution [4][5][6][7][8][9] offers an alternative method for improving the robustness of proteins in biotechnology, 10 with error-prone polymerase chain reaction (epPCR) and/or DNA shuffling being the most popular gene mutagenesis techniques in this endeavor. In many cases this approach is superior to rational design, although at the expense of having to screen large mutant libraries, which is still considered to be the bottleneck of directed evolution in general.…”

Section: Introductionmentioning

confidence: 99%

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Augustyniak

Brzezinska

Pijning³

et al. 2012

Protein Science

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Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

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Section: Introductionmentioning

confidence: 99%

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Augustyniak

Brzezinska

Pijning³

et al. 2012

Protein Science

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“…Early random mutagenesis strategies using radiation and chemical mutagens such as ethyl methanesulfonate have today been replaced by the use of error-prone DNA polymerases (17). The development of new fluorescent proteins (20) has made use of error-prone PCR (4) to generate mutant variants that can be screened by examining plates of Escherichia coli colonies under fluorescent illumination (3,16) or by fluorescence-activated cell sorting (2,18).…”

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confidence: 99%

Viral Mutagenesis as a Means for Generating Novel Proteins

Davis

Pol

2010

J Virol

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We demonstrate that a mutation-prone virus engineered to express a foreign gene is an expedient means for generating novel mutant nonviral proteins in mammalian cells. Using vesicular stomatitis virus to express a gene coding for a fluorescent DsRed protein, a number of green mutant variants including a new variant not previously described were rapidly isolated from infected cells, sequenced, and cloned. Similar methods may be useful in the development of physiologically sensitive fluorescent reporter proteins and directed evolution or mutagenesis of proteins in general.

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“…The process of directed evolution conventionally entails generating molecular diversity through random mutagenesis and in vitro recombination followed by high-throughput selection. [33,34] The need for effective selection markers for specific photocatalytic properties makes it challenging to apply this approach to engineering proteins for artificial photosynthesis. [35] Rational design, however, involves an engineering approach to protein design, often in combination with molecular modelling of the protein structure.…”

Section: Engineering Proteins For Artificial Photosynthesismentioning

confidence: 99%

Perspectives for Photobiology in Molecular Solar Fuels

Hingorani

Hillier

2012

Aust. J. Chem.

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This paper presents an overview of the prospects for bio-solar energy conversion. The Global Artificial Photosynthesis meeting at Lord Howe Island (14–18 August 2011) underscored the dependence that the world has placed on non-renewable energy supplies, particularly for transport fuels, and highlighted the potential of solar energy. Biology has used solar energy for free energy gain to drive chemical reactions for billions of years. The principal conduits for energy conversion on earth are photosynthetic reaction centres – but can they be harnessed, copied and emulated? In this communication, we initially discuss algal-based biofuels before investigating bio-inspired solar energy conversion in artificial and engineered systems. We show that the basic design and engineering principles for assembling photocatalytic proteins can be used to assemble nanocatalysts for solar fuel production.

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Advances in generating functional diversity for directed protein evolution

Cited by 154 publications

References 39 publications

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Viral Mutagenesis as a Means for Generating Novel Proteins

Perspectives for Photobiology in Molecular Solar Fuels

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