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

Scientific Reports
Tanja Weiffert, Sara Linse

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 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. Functio...Continue Reading

References

Aug 7, 1990·Biochemistry·K A Dill
Jan 1, 1990·Proteins·G J Lesser, G D Rose
Aug 1, 1989·Protein Engineering·S H KimT K Lee
Nov 26, 1987·Biochimica Et Biophysica Acta·R W WilliamsS Loughran
Jan 28, 1972·Biochimica Et Biophysica Acta·J A Morris, R H Cagan
Feb 1, 1995·Chemical Senses·J R SomozaS H Kim
Oct 25, 1994·Proceedings of the National Academy of Sciences of the United States of America·N Johnsson, A Varshavsky
Jan 1, 1993·Annual Review of Biophysics and Biomolecular Structure·G D Rose, R Wolfenden
Aug 5, 1997·Proceedings of the National Academy of Sciences of the United States of America·F RossiH M Blau
Aug 26, 1999·International Journal of Pharmaceutics·W Wang
Jan 12, 2001·Journal of Molecular Biology·R SpadacciniP A Temussi
Aug 18, 2001·Cell·G NelsonC S Zuker
Oct 16, 2001·Journal of Agricultural and Food Chemistry·T MasudaN Kitabatake
Mar 28, 2002·Proceedings of the National Academy of Sciences of the United States of America·Xiaodong LiElliot Adler
Sep 23, 2003·Protein Science : a Publication of the Protein Society·Ryan DayValerie Daggett
Apr 21, 2004·European Journal of Biochemistry·Yawen Bai, Hanqiao Feng
Jun 25, 2004·Bioscience, Biotechnology, and Biochemistry·Yukako ShirasukaKeiko Abe
Jul 9, 2004·Molecular & Cellular Proteomics : MCP·Andrei AlexandrovElizabeth J Grayhack
Sep 24, 2004·Journal of Biotechnology·Vincent G H EijsinkGert Vriend
Apr 29, 2005·Biomolecular Engineering·Vincent G H EijsinkBertus van den Burg
Aug 19, 2005·Journal of Medicinal Chemistry·Gabriella MoriniPiero A Temussi
Sep 9, 2005·Nature Methods·Christopher G M WilsonLynne Regan
Jun 13, 2006·Journal of Molecular Biology·Veronica EspositoPiero A Temussi
Nov 14, 2006·Nature Methods·Ingrid Remy, Stephen W Michnick
Nov 17, 2006·Nature·Jayaram ChandrashekarCharles S Zuker
Feb 22, 2007·Nucleic Acids Research·Christoph ScheichKonrad Büssow
May 22, 2007·Journal of Molecular Biology·Jie-rong HuangSophie E Jackson
Dec 23, 2008·Biochimica Et Biophysica Acta·Wei-Feng XueJannette Carey
May 28, 2009·Protein Science : a Publication of the Protein Society·Stina LindmanSara Linse
Mar 5, 2010·Biochemistry·Veronica EspositoPiero A Temussi
Nov 3, 2010·Proceedings of the National Academy of Sciences of the United States of America·Stina LindmanSara Linse
Nov 1, 2011·Journal of Molecular Recognition : JMR·Piero Andrea Temussi
May 23, 2014·FEBS Letters·C Nick PaceGerald R Grimsley
Oct 27, 2015·Current Opinion in Structural Biology·Thomas J Magliery
May 11, 2016·Journal of Computer-aided Molecular Design·Shweta KulshreshthaNidhi Mathur

❮ Previous
Next ❯

Citations

Jul 7, 2018·Chemical Communications : Chem Comm·Mattias TörnquistSara Linse

❮ Previous
Next ❯

Methods Mentioned

BETA
glycosylation
phage display
PCR
gel filtration
electrophoresis
NMR
circular dichroism
fluorescence-activated cell sorting

Software Mentioned

Pymol

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.