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Protein stabilization with retained function of monellin using a split GFP system

Weiffert, Tanja LU and Linse, Sara LU (2018) In Scientific Reports 8(1).
Abstract

Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein monellin, with increased stability, were derived by an in vivo screening method based on the thermodynamic linkage between fragment complementation and protein stability. This approach depends on the correlation between mutational effects on the affinity between protein fragments and the stability of the intact protein. By linking the two fragments of monellin to the split GFP (green fluorescent protein) system, reconstitution of GFP was promoted and moderately fluorescent colonies were obtained. Two separate random libraries were produced for the monellin chains and... (More)

Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein monellin, with increased stability, were derived by an in vivo screening method based on the thermodynamic linkage between fragment complementation and protein stability. This approach depends on the correlation between mutational effects on the affinity between protein fragments and the stability of the intact protein. By linking the two fragments of monellin to the split GFP (green fluorescent protein) system, reconstitution of GFP was promoted and moderately fluorescent colonies were obtained. Two separate random libraries were produced for the monellin chains and the mutant clones were ranked based on fluorescence intensity. Mutants with increased affinity between the fragments, and subsequently increased stability, caused increased fluorescence intensity of split GFP. Single chain monellin variants of the top-ranked mutants for each chain, S76Y in the A-chain and W3C + R39G in the B-chain and all combinations thereof, were expressed and the increase in stability was verified by temperature denaturation studies using circular dichroism spectroscopy. Functionality studies showed that mutant S76Y has retained sweetness and has potential use within the food industry.

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type
Contribution to journal
publication status
published
subject
in
Scientific Reports
volume
8
issue
1
article number
12763
publisher
Nature Publishing Group
external identifiers
  • scopus:85052239947
  • pmid:30143736
ISSN
2045-2322
DOI
10.1038/s41598-018-31177-z
language
English
LU publication?
yes
id
7db296da-09b6-4730-96cb-182f3f9c4fde
date added to LUP
2018-09-24 13:57:24
date last changed
2021-09-15 02:48:52
@article{7db296da-09b6-4730-96cb-182f3f9c4fde,
  abstract     = {<p>Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein monellin, with increased stability, were derived by an in vivo screening method based on the thermodynamic linkage between fragment complementation and protein stability. This approach depends on the correlation between mutational effects on the affinity between protein fragments and the stability of the intact protein. By linking the two fragments of monellin to the split GFP (green fluorescent protein) system, reconstitution of GFP was promoted and moderately fluorescent colonies were obtained. Two separate random libraries were produced for the monellin chains and the mutant clones were ranked based on fluorescence intensity. Mutants with increased affinity between the fragments, and subsequently increased stability, caused increased fluorescence intensity of split GFP. Single chain monellin variants of the top-ranked mutants for each chain, S76Y in the A-chain and W3C + R39G in the B-chain and all combinations thereof, were expressed and the increase in stability was verified by temperature denaturation studies using circular dichroism spectroscopy. Functionality studies showed that mutant S76Y has retained sweetness and has potential use within the food industry.</p>},
  author       = {Weiffert, Tanja and Linse, Sara},
  issn         = {2045-2322},
  language     = {eng},
  month        = {08},
  number       = {1},
  publisher    = {Nature Publishing Group},
  series       = {Scientific Reports},
  title        = {Protein stabilization with retained function of monellin using a split GFP system},
  url          = {http://dx.doi.org/10.1038/s41598-018-31177-z},
  doi          = {10.1038/s41598-018-31177-z},
  volume       = {8},
  year         = {2018},
}