Avidin engineering : modification of function, oligomerization, stability and structure topology
Julkaistu sarjassa
Jyväskylä studies in biological and environmental scienceTekijät
Päivämäärä
2003Oppiaine
MolekyylibiologiaAvidiini on neljästä samanlaisesta alayksiköstä muodostunut tetrameerinen glykoproteiini, jota esiintyy luontaisesti, pieninä pitoisuuksina, kanan munanvalkuaisessa. Eräs avidiinin merkittävimmistä ominaisuuksista on sen kyky sitoa vitamiini biotiinia. Avidiinin ja biotiinin välinen vuorovaikutus on voimakkain tunnettu luonnossa esiintyvä ei-kovalenttinen sidos proteiinin ja sen ligandin välillä. Tätä ominaisuutta on hyödynnetty jo pitkään lukuisissa bio- ja lääketieteellisissä sovelluksissa, jotka yhdessä muodostavat niin kutsutun (strept)avidiini-biotiini -teknologian. Avidin is a basic and rather stable tetrameric glycoprotein found in chicken egg-white. It has an enormous affinity toward biotin (vitamin H), which forms the basis of so called avidin-biotin technology. This extraordinary protein-ligand pair is superior to many other protein-ligand systems because it is very robust and can be used under even harsh experimental conditions. In addition, several biotin derivatives and conjugates are commercially available. Our main objective was to create new avidin tools with novel properties for avidin-biotin technology which would have improved performance in some applications and even open completely new possibilities in others. In addition we obtained valuable basic information about avidin. We modified avidin by the means of protein engineering using a rational mutation design strategy. We succeeded in engineering an avidin with enhanced thermal stability characteristics by introducing cysteine residues to the monomer-monomer interfaces, which formed covalent intermonomeric disulfide bridges. We showed also that the tetrameric quaternary structure of avidin can be broken by only two crucial point mutations in the interface residues and that the resultant monomeric avidin was biologically active in the sense of biotin binding. In addition, we prepared two different circularly permuted avidins and their fusion. The resultant pseudo-tetrameric fusion avidin, in which half of the binding sites can be further modified independently of the other half, has enormous potential in new applications. Finally, we introduced histidine residues into the monomer-monomer interfaces, which led to some of the mutants becoming pH dependent with respect to biotin binding and oligomerization.
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Julkaisija
Jyväskylän yliopistoISBN
951-39-1650-2ISSN Hae Julkaisufoorumista
1456-9701Asiasanat
Metadata
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- Väitöskirjat [3559]
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