CHOWDHURY, ANANDA
(2014)
FUNCTIONAL HUMAN CANNABINOID RECEPTOR 2 FROM ESCHERICHIA COLI.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
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Abstract
The Cannabinoid Receptor 2 (CB2), a member of the endocannabinoid system belongs to the Rhodopsin family of G-Protein Coupled Receptors (GPCRs). It is expressed mainly in the immune cells and exerts immunomodulatory roles in normal and pathophysiological conditions. Therapeutic modulation of the CB2 presents a promising strategy for the treatment of several diseases like multiple myeloma, osteoporosis, pain etc. In the face of the huge therapeutic importance of the CB2, high resolution structural information and mechanistic details of receptor activation are poorly understood. This principally owes to the paucity of large amounts of purified recombinant functionally active CB2 in-vitro. GPCRs and most eukaryotic membrane proteins pose a formidable challenge for recombinant expression and purification. Limitations include low expression, toxicity towards host cells, loss of function etc. In an effort to produce functionally active recombinant CB2 that can be used for subsequent structural studies, in the present study, we have developed two distinct approaches for the functional expression and purification of CB2 from the E. coli.
In the first approach we used Mistic, an integral membrane protein expression enhancer, and TarCF, a C-terminal fragment of the bacterial chemosensory transducer Tar, as fusion partners at the N'- and C'-terminal respectively of the CB2 for its membrane targeted expression in the E. coli C43(DE3). Using the fusion partners individually or in combination, we found that CB2 fusion protein expression was maximal when both partners were used in combination. More importantly, the fusion protein Mistic–CB2–TarCF localized to the E. coli membrane and these extracted membrane fractions exhibited functional binding activities with known CB2 ligands including CP55,940, WIN55,212-2 and SR144,528.
In the second approach, we expressed the CB2, in fusion with GST at its N'- terminal, as inactive inclusion bodies (IBs). The receptor protein was engineered to carry a 6 Histidine (His6) tag at its C'-terminal for subsequent immobilized metal affinity chromatographic (IMAC) purification. Pilot studies supported extraction of GST-CB2 in a denaturing detergent, N- Lauroyl sarcosinate (Sarkosyl) followed by exchange to Dodecyl-beta-D-Maltoside (DDM) for “on-column” cleavage. Post size exclusion chromatography, eluted purified monodisperse CB2 were subjected to refolding either in lipidic (DMPC) or proteic (Amphipol) environments. CB2 refolded in DMPC exhibited functional binding activities with known CB2 ligands including CP 55,940, SR144528 and PY2-64.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
13 January 2014 |
| Date Type: |
Publication |
| Defense Date: |
25 November 2013 |
| Approval Date: |
13 January 2014 |
| Submission Date: |
29 December 2013 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
174 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Pharmacy > Pharmaceutical Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Cannabinoid Receptor 2, GPCR, expression-purification, in-vitro refolding, functional assay |
| Date Deposited: |
13 Jan 2014 13:23 |
| Last Modified: |
15 Nov 2016 14:16 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/20327 |
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