Protein evolution via amino acid and codon elimination.

<h4>Background</h4>Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is...

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Autores principales:	Lise Goltermann, Marie Sofie Yoo Larsen, Rajat Banerjee, Andreas C Joerger, Michael Ibba, Thomas Bentin
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4e1500bbb47049579e98289bd008f2ef
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spelling	oai:doaj.org-article:4e1500bbb47049579e98289bd008f2ef2021-12-02T20:22:09ZProtein evolution via amino acid and codon elimination.1932-620310.1371/journal.pone.0010104https://doaj.org/article/4e1500bbb47049579e98289bd008f2ef2010-04-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20436666/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.<h4>Methodology/principal findings</h4>The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance.<h4>Conclusions/significance</h4>The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.Lise GoltermannMarie Sofie Yoo LarsenRajat BanerjeeAndreas C JoergerMichael IbbaThomas BentinPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 4, p e10104 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Lise Goltermann Marie Sofie Yoo Larsen Rajat Banerjee Andreas C Joerger Michael Ibba Thomas Bentin Protein evolution via amino acid and codon elimination.
description	<h4>Background</h4>Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.<h4>Methodology/principal findings</h4>The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance.<h4>Conclusions/significance</h4>The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.
format	article
author	Lise Goltermann Marie Sofie Yoo Larsen Rajat Banerjee Andreas C Joerger Michael Ibba Thomas Bentin
author_facet	Lise Goltermann Marie Sofie Yoo Larsen Rajat Banerjee Andreas C Joerger Michael Ibba Thomas Bentin
author_sort	Lise Goltermann
title	Protein evolution via amino acid and codon elimination.
title_short	Protein evolution via amino acid and codon elimination.
title_full	Protein evolution via amino acid and codon elimination.
title_fullStr	Protein evolution via amino acid and codon elimination.
title_full_unstemmed	Protein evolution via amino acid and codon elimination.
title_sort	protein evolution via amino acid and codon elimination.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/4e1500bbb47049579e98289bd008f2ef
work_keys_str_mv	AT lisegoltermann proteinevolutionviaaminoacidandcodonelimination AT mariesofieyoolarsen proteinevolutionviaaminoacidandcodonelimination AT rajatbanerjee proteinevolutionviaaminoacidandcodonelimination AT andreascjoerger proteinevolutionviaaminoacidandcodonelimination AT michaelibba proteinevolutionviaaminoacidandcodonelimination AT thomasbentin proteinevolutionviaaminoacidandcodonelimination
_version_	1718374106607386624

Protein evolution via amino acid and codon elimination.

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