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Immobilizing strategies for membranes to screen against antibody mimetics with phage display

Agboh, Vincent Ochejeni (2015) Immobilizing strategies for membranes to screen against antibody mimetics with phage display. PhD thesis, University of Leeds.

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Antibody mimetics are a novel class of potential therapeutic agents which improve on several limitations which hinder antibodies. As a result, they are the subject of increasing interest from both the pharmaceutical and academic sectors. They are commonly selected via phage display against an immobilised target. However the presentation of these targets, particularly in the case of membrane proteins, is often a rate limiting step in their production. In this study, several commonly-used protein tags were compared in their ability to immobilise a model membrane protein - MPSIL0294, a metal ion transporter from Enterococcus faecalis, a streptavidin binding peptide tag, an Avitag which has undergone in vivo biotinylation and a chemically biotinylated C-terminal cysteine. A fourth method of immobilisation was also included, aspecific chemical biotinylation of lysine residues in AcrB, a component of an E.coli multidrug efflux pump. The tags were compared using a phage display assay, in which designed ankyrin repeat proteins (DARPins) from a naïve library were screened against both model membrane proteins, which were both solubilised in n-Dodecyl-β-D-maltopyranoside (DDM). With the exception of the C-terminal cysteine variant of MPSIL0294, no DARPins capable of binding could be selected by any of the other immobilisation techniques after two rounds of phage display. Furthermore, the C-terminal cysteine MPSIL0294 also yielded poor success rates and only two binding DARPins were identified. Despite showing a moderately high level of sequence similarity, these two DARPins showed different binding activities during validation experiments. The alternative tags under test however; do not appear to be beneficial in the selection of antibody mimetics against immobilised membrane protein targets. Different methods of protein solubilisation were also compared in an identical manner: detergent solubilisation (in DDM), lipid nanodiscs and styrene maleic acid lipid particles (SMALP). These solubilisation methods were compared using a panel of membrane proteins composed of MPSIL0294, AcrB, NupC, an E.coli concentrative nucleoside transporter and VcCNT, NupCs homologue from Vibrio Cholerae. SMALPs of AcrB, VcCNT and NupC displayed the highest success rate for selecting DARPins capable of binding the respective targets. Subsequent validation tests showed that the best DARPins selected against AcrB SMALPs and nanodiscs behaved in a similar manner. SMALPs are relatively easy to produce, especially compared to nanodiscs so this study concludes that SMALPs are the best format for phage display.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
The University of Leeds > University of Leeds Research Centres and Institutes > Astbury Centre for Structural Molecular Biology (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute of Membrane and Systems Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.684525
Depositing User: Mr. Vincent Ochejeni Agboh
Date Deposited: 04 May 2016 11:39
Last Modified: 06 Oct 2016 14:42
URI: http://etheses.whiterose.ac.uk/id/eprint/12705

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