Expression and purification of truncated, non-glycosylated turkey beta-adrenergic receptors for crystallization |
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Authors: | Tony Warne |
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Affiliation: | MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK |
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Abstract: | In order to purify milligram quantities of turkey β-adrenergic receptor (βAR) for structural analysis, we have expressed mutant βARs using the baculovirus system. The initial βAR construct was truncated at both N- and C-termini thus removing an N-glycosylation site. Cys 116 was mutated to leucine and a histidine tag was added at the C-terminus resulting in the βAR construct 20-424/His6. Expression of this construct in Sf9 cells produced 0.5 mg of unpurified receptor per liter of culture which necessitated the use of a fermenter for large-scale production. The yield was improved more than 2-fold to 1.2 mg/l culture by using Tni cells which facilitated the production of receptor on a 4 litre scale in shake cultures. The receptor was purified to homogeneity with 35% recovery giving a yield of 2 mg receptor. A further deletion at the N-terminus (βAR 34-424/His6) eliminated proteolysis which had been observed with the original construct and also increased expression more than 5-fold to 360 pmol/mg solubilized membrane protein. This expression level is one of the highest reported for a G protein-coupled receptor (GPCR) and has enabled us to purify 10 mg βAR for large-scale crystallization experiments. |
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Keywords: | β-adrenergic receptor Expression Purification |
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