Type
Text
Type
Thesis
Advisor
Brown, Deborah | French, Jarrod.
Date
2015-12-01
Keywords
Biochemistry | caveolae, cavin-1, protein purification
Department
Department of Biochemistry and Cell Biology.
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/76930
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Caveolae are nanoscale invaginations found in the plasma membrane of mammalian cells, and are implicated in numerous essential cellular communication and transport processes. They are characterized by a coat composed of caveolin and cavin proteins. Recent studies of the caveolar coat complex suggest that cavin-1 associates with caveolin-1 during caveolae biogenesis. However, it is not known how this binding occurs. Studies of the spatial and physical interactions between cavin-1 and caveolin-1 can provide valuable insights into the mechanism of caveolae generation. The goal of this study was to test two different fusion tags to purify a sufficient quantity of cavin-1, for use in future binding studies with caveolin-1. We successfully generated a plasmid encoding 6His-SUMO-cavin-1 by cloning full length cavin-1 into the LIC SUMO vector 2S-T, and expressed it in BL21 (DE3) pLysS. Parameters of temperature, concentration of IPTG, detergent type, and length of incubation after IPTG-induction, were tested to optimize cavin-1 expression conditions. Immobilized metal affinity chromatography with Ni-NTA resin tested binding and release of the SUMO-tagged cavin-1. The poor protein yields from tests on 6-His-SUMO-cavin-1 underscored persistent issues of solubility, suboptimal binding to the resin, and inadequate release of purified protein. Studies on an earlier construct, GST-cavin-1, were then resumed to evaluate and maximize binding and release of GST-cavin-1 from glutathione-agarose beads. While inefficient bead binding problems remained, GST-cavin-1 yielded a more consistent recovery of the desired protein. As results suggested that enough soluble GST-cavin-1 could be obtained for binding studies, we performed a large-scale preparation of the protein for use in a diagnostic test for association with caveolin-1. We did not observe any binding in this preliminary test. The results prompted further questions about the stoichiometry of the cavin-1 and caveolin-1 complex, and highlighted the possibility of additional layers of complexity involved in this interaction. | 77 pages
Recommended Citation
Siddiqui, Safa Farjand, "Developing Tools for Cavin-1 Purification and Binding Studies with Caveolin-1" (2015). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 2803.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/2803