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Title: Studies on the STE6-encoded a-factor pump of the yeast Saccharomyces cerevisiae
Author: Brown, Ann Melanie
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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The Saccharomyces cerevisiae STE6 gene product mediates the export of the peptide mating pheromone a-factor. The Ste6 polypeptide (Ste6p) belongs to the ABC transporter superfamily whose members include many proteins of medical significance. Ste6p is a short-lived transmembrane protein which is produced in low levels in wild-type yeast. This thesis describes the construction, expression and partial purification of a recombinant form of Ste6p from S. cerevisiae. In the absence of a functional assay for Ste6p its presence was detected by Western blot analysis using polyclonal antibodies raised in this study. The antibodies were produced in rabbits immunised with a recombinant Ste6p-ProteinA fusion protein. Purification of wild-type Ste6p was hindered by the very low levels at which the protein was being produced. As an alternative to conventional purification techniques, Ste6p was affinity-tagged at its extreme N-terminus with a six histidine residue (to produce N(His)6Ste6p) so that it could be absorbed from a dilute solution by its high affinity to Ni-NTA (nickel-Nitrilo-Tri-Acetic-acid) resin. The chimaeric protein was expressed under the control of the GAL promoter in a MATa, protease deficient strain of S. cerevisiae against a background of wild-type protein. Purification of N(His)6Ste6p failed due to an apparent inability of the chimaeric protein to bind to the resin. Extracts of cells expressing N(His)6Ste6p were Western blotted and probed with an anti-histidine-tag monoclonal antibody. The antibody failed to detect any protein of the correct size for the Ste6p chimaera. These results suggested that the N-terminus of N(His)6Ste6p had been removed during post-translational modification of the protein. The third approach to the purification of Ste6p involved tagging the C-terminus of the protein with eight histidine residues to produce C(His)8Ste6p. As with the N-terminally tagged Ste6p, this chimaera was expressed under the control of the GAL promoter, however in this case the protein was expressed in a MATαpep4 strain of S. cerevisae. This protein could be detected by the anti-histidine tag monoclonal antibody and was able to bind to the Ni-NTA resin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available