Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486907
Title: Expression, purification and structural analysis of bacterial ABC transporters and v-atpase subunits
Author: Gutmann, Daniel A. P.
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2009
Availability of Full Text:
Full text unavailable from EThOS.
Please contact the current institution’s library for further details.
Abstract:
The aim of the project was to produce stable, soluble multidrug resistance (mdr) ABC. transporters and V-type ATPase subunits suitable for structural studies in order to gain insight into these two families of ATP metabolising membrane proteins. Mdr ABC transporters from a number 'of bacteria were successfully expressed. The best protein in terms of yield of pure protein (5mg.r1) was LmrA from LactococQus factis. Due to seripus aggregation this protein resisted all initial attempts at crystallisation. A novel method was developed, the Ultracentrifugation dispersity sedimentation (UDS) assay, to allow screening a wide range of buffer and detergent conditions to limit the aggregation problem. A small of conditions was identified and LmrA re-submitted to crystallisation trials. Preliminary crystals of LmrA were obtained in the detergents n-dodecylfoscholine (FC-12) and polyoxyethylene(8)dodecyl ether (C12ES). The �·UDS assay has also been used successfully to screen detergents for a range of other membrane proteins. Both the E and G subunits of V-type ATPase from T. thermophilus have been overexpressed and purified. In addition, a method for the reconstitution of the EG complex developed and optimised to produce amounts sufficient for crystallisation trials. Initial crystallisation screens identified several crystallisation leads for subunit E and the EG complex that were further optimised to oqtain diffraction quality crystals. Eventually, both subunit E and the EG complex have yielded diffracting crystals to 3.4 Aand 4.0 Aresolution, respectively. Further work will focus on obtaining better diffracting crystals and phasing of the data.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.486907  DOI: Not available
Share: