Expression and purification of truncated, non-glycosylated turkey beta-adrenergic receptors for crystallization.
In order to purify milligram quantities of turkey beta-adrenergic receptor (betaAR) for structural analysis, we have expressed mutant betaARs using the baculovirus system. The initial betaAR 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 betaAR 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 (betaAR 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 betaAR for large-scale crystallization experiments.
Warne
Tony
T
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. ajw2@mrc-lmb.cam.ac.uk
Chirnside
Jill
J
Schertler
Gebhard F X
GF
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003460:Crystallization; D003470:Culture Media; D005285:Fermentation; D017353:Gene Deletion; D007313:Insecta; D008969:Molecular Sequence Data; D011943:Receptors, Adrenergic, beta / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D043562:Receptors, G-Protein-Coupled; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014422:Turkeys; D015854:Up-Regulation
D003470:Culture Media; C109334:GPR1 protein, S cerevisiae; D011943:Receptors, Adrenergic, beta; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D029701:Saccharomyces cerevisiae Proteins