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Mutations in maltose-binding protein that alter affinity and solubility properties

Maltose-binding protein (MBP) from Escherichia coli has been shown to be a good substrate for protein engineering leading to altered binding (Marvin and Hellinga, Proc Natl Acad Sci U S A 98:4955–4960, 2001a) and increased affinity (Marvin and Hellinga, Nat Struct Biol 8:795–798, 2001b; Telmer and S...

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Autores principales: Walker, Iris H., Hsieh, Pei-chung, Riggs, Paul D.
Formato: Texto
Lenguaje:English
Publicado: Springer-Verlag 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2940430/
https://www.ncbi.nlm.nih.gov/pubmed/20535468
http://dx.doi.org/10.1007/s00253-010-2696-y
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author Walker, Iris H.
Hsieh, Pei-chung
Riggs, Paul D.
author_facet Walker, Iris H.
Hsieh, Pei-chung
Riggs, Paul D.
author_sort Walker, Iris H.
collection PubMed
description Maltose-binding protein (MBP) from Escherichia coli has been shown to be a good substrate for protein engineering leading to altered binding (Marvin and Hellinga, Proc Natl Acad Sci U S A 98:4955–4960, 2001a) and increased affinity (Marvin and Hellinga, Nat Struct Biol 8:795–798, 2001b; Telmer and Shilton, J Biol Chem 278:34555–34567, 2003). It is also used in recombinant protein expression as both an affinity tag and a solubility tag. We isolated mutations in MBP that enhance binding to maltodextrins 1.3 to 15-fold, using random mutagenesis followed by screening for enhanced yield in a microplate-based affinity purification. We tested the mutations for their ability to enhance the yield of a fusion protein that binds poorly to immobilized amylose and their ability to enhance the solubility of one or more aggregation-prone recombinant proteins. We also measured dissociation constants of the mutant MBPs that retain the solubility-enhancing properties of MBP and combined two of the mutations to produce an MBP with a dissociation constant 10-fold tighter than wild-type MBP. Some of the mutations we obtained can be rationalized based on the previous work, while others indicate new ways in which the function of MBP can be modified. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-010-2696-y) contains supplementary material, which is available to authorized users.
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spelling pubmed-29404302010-10-04 Mutations in maltose-binding protein that alter affinity and solubility properties Walker, Iris H. Hsieh, Pei-chung Riggs, Paul D. Appl Microbiol Biotechnol Applied Genetics and Molecular Biotechnology Maltose-binding protein (MBP) from Escherichia coli has been shown to be a good substrate for protein engineering leading to altered binding (Marvin and Hellinga, Proc Natl Acad Sci U S A 98:4955–4960, 2001a) and increased affinity (Marvin and Hellinga, Nat Struct Biol 8:795–798, 2001b; Telmer and Shilton, J Biol Chem 278:34555–34567, 2003). It is also used in recombinant protein expression as both an affinity tag and a solubility tag. We isolated mutations in MBP that enhance binding to maltodextrins 1.3 to 15-fold, using random mutagenesis followed by screening for enhanced yield in a microplate-based affinity purification. We tested the mutations for their ability to enhance the yield of a fusion protein that binds poorly to immobilized amylose and their ability to enhance the solubility of one or more aggregation-prone recombinant proteins. We also measured dissociation constants of the mutant MBPs that retain the solubility-enhancing properties of MBP and combined two of the mutations to produce an MBP with a dissociation constant 10-fold tighter than wild-type MBP. Some of the mutations we obtained can be rationalized based on the previous work, while others indicate new ways in which the function of MBP can be modified. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-010-2696-y) contains supplementary material, which is available to authorized users. Springer-Verlag 2010-06-10 2010 /pmc/articles/PMC2940430/ /pubmed/20535468 http://dx.doi.org/10.1007/s00253-010-2696-y Text en © The Author(s) 2010 https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
spellingShingle Applied Genetics and Molecular Biotechnology
Walker, Iris H.
Hsieh, Pei-chung
Riggs, Paul D.
Mutations in maltose-binding protein that alter affinity and solubility properties
title Mutations in maltose-binding protein that alter affinity and solubility properties
title_full Mutations in maltose-binding protein that alter affinity and solubility properties
title_fullStr Mutations in maltose-binding protein that alter affinity and solubility properties
title_full_unstemmed Mutations in maltose-binding protein that alter affinity and solubility properties
title_short Mutations in maltose-binding protein that alter affinity and solubility properties
title_sort mutations in maltose-binding protein that alter affinity and solubility properties
topic Applied Genetics and Molecular Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2940430/
https://www.ncbi.nlm.nih.gov/pubmed/20535468
http://dx.doi.org/10.1007/s00253-010-2696-y
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