NDLI: The synthesis and high-level expression of a β2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line

Content Provider	PubMed Central
Author	Chelikani, Prashen Reeves, Philip J. Rajbhandary, Uttam L. Khorana, H. Gobind
Abstract	High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure–function studies. The main goal of the present work was to improve expression levels of β2-adrenergic receptor (β2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster β2-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 ± 3 pmol β2-AR/mg of membrane protein, as measured by saturation binding assay using the β2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of β2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type β2-AR at levels of 220 ± 40 pmol/mg of membrane protein corresponding to 50 ± 8 μg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 ± 3 μg/15-cm plate. This level of expression now makes it feasible to pursue structure–function studies using EPR. Furthermore, scale-up of β2-AR expression using suspension cultures in a bioreactor should now allow production of enough β2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.
Related Links	http://dx.doi.org/10.1110/ps.062080006
Starting Page	1433
File Format	PDF
ISSN	1469896X
e-ISSN	1469896X
Journal	Protein Science : A Publication of the Protein Society
Issue Number	6
Volume Number	15
Language	English
Publisher	Cold Spring Harbor Laboratory Press
Publisher Date	2006-06-01
Access Restriction	Open
Rights Holder	Cold Spring Harbor Laboratory Press
Subject Keyword	Proteomics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
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7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
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9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

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