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Expression and purification of membrane proteins: Focus on the G-protein coupled receptor MC4r

Dolby, Viveka LU (2004)
Abstract (Swedish)
Popular Abstract in Swedish

10.SWEDISH SUMMARY(Populärvetenskaplig sammanfattning) Proteiner är viktiga byggstenar i cellen och är uppbyggda av kroppens tjugo olika aminosyror. Varje protein har en unik egenskap och spelar en viktig roll för cellens funktion. Vissa proteiner löser sig i vatten (hydrofila) medan andra är olösliga (hydrofoba). Membranproteiner är hydrofoba proteiner som är inbäddade i cellens membran. Dessa består av lipider och avgränsar celler och cellens olika delar. Membranproteiner deltager i otaliga funktioner i kroppen. Därför är det speciellt viktigt att få större kunskap om dessa proteiner för att kunna göra framsteg inom läkemedelsindustrin och utveckla nya läkemedel. Denna avhandling behandlar två... (More)
Popular Abstract in Swedish

10.SWEDISH SUMMARY(Populärvetenskaplig sammanfattning) Proteiner är viktiga byggstenar i cellen och är uppbyggda av kroppens tjugo olika aminosyror. Varje protein har en unik egenskap och spelar en viktig roll för cellens funktion. Vissa proteiner löser sig i vatten (hydrofila) medan andra är olösliga (hydrofoba). Membranproteiner är hydrofoba proteiner som är inbäddade i cellens membran. Dessa består av lipider och avgränsar celler och cellens olika delar. Membranproteiner deltager i otaliga funktioner i kroppen. Därför är det speciellt viktigt att få större kunskap om dessa proteiner för att kunna göra framsteg inom läkemedelsindustrin och utveckla nya läkemedel. Denna avhandling behandlar två olika membranproteiner som finns i kroppen, melanocortin 4 receptorn (MC4r) och enzymet 11 b-hydroxysteroid dehydrogenase typ 1 (11b-HSD1). För att studera proteiner utanför människokroppen brukar dessa tillverkas i en annan värdorganism. Genom att sätta in genen, som kodar för proteinet, hos en annan organism kommer denna att producera proteinet. Detta kallas att man uttrycker proteinet rekombinant. De rekombinanta proteinerna isoleras och karaktäriseras för att studera deras biologiska funktion. För att kunna isolera hydrofoba proteiner från cellen används detergenter vilka är amfifila, det vill säga de har en hydrofil och en hydrofob del. Vid tillsats av detergenter delas cellmembranen upp och protein-detergent komplex bildas. Detta kallas solubilisering. Protein-detergent komplexen är hydrofila och håller sig i lösning. Efter solubilisering kan upprening av specifika membranproteiner utföras med hjälp av separationsmetoder där kontaminanter tas bort. I avhandlingen har kromatografi och vattenhaltiga två-fas system använts för upprening av membranproteiner. I avhandlingens två första artiklar studeras uttrycket av MC4r i mammalieceller och insektsceller. Studierna visar att det är möjligt att uttrycka MC4r i de olika organismerna. Genom att aktivera proteinet med olika bindande molekyler kan man karaktärisera receptorn. I artiklarna visas att MC4r kan förmedla signaler över membranet till ett aktiveringsprotein, som kallas G-protein, för vidare signalering i cellen. I artikel tre beskrivs en ny metod för isolering av membranproteiner som bygger på kombination av vattenhaltiga detergent/polymer system och affinitetsmatris. Metoden tillämpades på upprening av 11 b-HSD 1 och visades vara mer effektiv än affinitetskromatografi. I artikel fyra har en isolering gjorts av MC4r genom att använda olika kromatografimetoder. Resultaten visar att det går att isolera proteinet och avlägsna alla kontaminanter. Dessa studier är viktiga för kunskap om membranproteiner, vilket kan leda till att nya läkemedel utvecklas. (Less)
Abstract
Membrane proteins are crucial components of the cell and are involved in many biological processes. Recombinant overexpression systems together with different purification methods are necessary to obtain large amounts of purified receptor for biophysical and functional studies. More knowledge about membrane proteins, and especially G-protein coupled receptors, would facilitate the development of imporatant future drug candidates. Intrinsic membrane proteins are embedded in the membrane and detergents are used to extract them from the membrane prior to purification. It is important to perform solubilisation with suitable detergents in order to prevent aggregation and denaturation. This thesis presents results from the study of two membrane... (More)
Membrane proteins are crucial components of the cell and are involved in many biological processes. Recombinant overexpression systems together with different purification methods are necessary to obtain large amounts of purified receptor for biophysical and functional studies. More knowledge about membrane proteins, and especially G-protein coupled receptors, would facilitate the development of imporatant future drug candidates. Intrinsic membrane proteins are embedded in the membrane and detergents are used to extract them from the membrane prior to purification. It is important to perform solubilisation with suitable detergents in order to prevent aggregation and denaturation. This thesis presents results from the study of two membrane proteins; the human melanocortin 4 receptor and the human membrane bound enzyme 11beta hydroxysteroid dehydrogenase type 1 (11beta-HSD 1). The MC4r is a G-protein coupled receptor with seven transmembrane regions, which mediates signalling to a G-protein upon receptor activation. The G-protein, activated by MC4r, is able to stimulate adenylate cyclase, and thus stimulate the formation of cAMP. The 11beta-HSD 1 is a membrane bound enzyme in the endoplasmatic reticulum. The enzyme contains one transmembrane region and the protein is a NADP(H) dependent enzyme and is responsible for the reversible interconversion of active cortisol to inactive cortisone.



The results present overexpression of MC4r in mammalian CHO cells and His-tagged MC4r in insect cells using the baculovirus infection system. The enzyme 11 beta-HSD 1 was succesfully overexpressed in yeast cells. Purification strategies were developed for the target proteins in order to obtain enriched and pure fractions of the proteins. Both MC4r and the 11 beta-HSD1 were expressed with histidine tags to enhance purification. The tagged MC4r was successfully purified using two-affinity steps (Ni-NTA and Heparin) together with an ion-exchange chromatography step. The enzyme 11 beta-HSD 1 was efficiently purified in a detergent/polymer system (Dextran 500/Tween 20) in combination with affinity resin. Most of the work was performed on the MC4 receptor, and is therefore the main focus of the thesis. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Dr Bill, Roslyn M, Aston University, Birmingham, UK
organization
publishing date
type
Thesis
publication status
published
subject
keywords
enzymology, Proteiner, enzymologi, Proteins, melanocortin 4 receptor, GPCR, purification, Expression, membrane proteins
pages
115 pages
publisher
Viveka Dolby, Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund,
defense location
Center for Chemistry and Chemical Engineering, Room B
defense date
2004-10-22 10:15
ISBN
91-7422-058-6
language
English
LU publication?
yes
id
4d7d1b32-57ff-4a52-a15d-d5e944066438 (old id 467339)
date added to LUP
2007-09-25 20:35:03
date last changed
2016-09-19 08:45:03
@misc{4d7d1b32-57ff-4a52-a15d-d5e944066438,
  abstract     = {Membrane proteins are crucial components of the cell and are involved in many biological processes. Recombinant overexpression systems together with different purification methods are necessary to obtain large amounts of purified receptor for biophysical and functional studies. More knowledge about membrane proteins, and especially G-protein coupled receptors, would facilitate the development of imporatant future drug candidates. Intrinsic membrane proteins are embedded in the membrane and detergents are used to extract them from the membrane prior to purification. It is important to perform solubilisation with suitable detergents in order to prevent aggregation and denaturation. This thesis presents results from the study of two membrane proteins; the human melanocortin 4 receptor and the human membrane bound enzyme 11beta hydroxysteroid dehydrogenase type 1 (11beta-HSD 1). The MC4r is a G-protein coupled receptor with seven transmembrane regions, which mediates signalling to a G-protein upon receptor activation. The G-protein, activated by MC4r, is able to stimulate adenylate cyclase, and thus stimulate the formation of cAMP. The 11beta-HSD 1 is a membrane bound enzyme in the endoplasmatic reticulum. The enzyme contains one transmembrane region and the protein is a NADP(H) dependent enzyme and is responsible for the reversible interconversion of active cortisol to inactive cortisone.<br/><br>
<br/><br>
The results present overexpression of MC4r in mammalian CHO cells and His-tagged MC4r in insect cells using the baculovirus infection system. The enzyme 11 beta-HSD 1 was succesfully overexpressed in yeast cells. Purification strategies were developed for the target proteins in order to obtain enriched and pure fractions of the proteins. Both MC4r and the 11 beta-HSD1 were expressed with histidine tags to enhance purification. The tagged MC4r was successfully purified using two-affinity steps (Ni-NTA and Heparin) together with an ion-exchange chromatography step. The enzyme 11 beta-HSD 1 was efficiently purified in a detergent/polymer system (Dextran 500/Tween 20) in combination with affinity resin. Most of the work was performed on the MC4 receptor, and is therefore the main focus of the thesis.},
  author       = {Dolby, Viveka},
  isbn         = {91-7422-058-6},
  keyword      = {enzymology,Proteiner,enzymologi,Proteins,melanocortin 4 receptor,GPCR,purification,Expression,membrane proteins},
  language     = {eng},
  pages        = {115},
  publisher    = {ARRAY(0x9c3a318)},
  title        = {Expression and purification of membrane proteins: Focus on the G-protein coupled receptor MC4r},
  year         = {2004},
}