Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors.
In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1mg/ml of reaction volume for 6-8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.
Ishihara
Goshi
G
RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Goto
Mie
M
Saeki
Mihoro
M
Ito
Kaori
K
Hori
Tetsuya
T
Kigawa
Takanori
T
Shirouzu
Mikako
M
Yokoyama
Shigeyuki
S
D000818:Animals; D002474:Cell-Free System; D004247:DNA / Q000235:genetics; D003902:Detergents; D004072:Digitonin; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008561:Membrane Fusion; D000077423:Polidocanol; D011092:Polyethylene Glycols; D014176:Protein Biosynthesis; D051381:Rats; D043585:Receptor, Muscarinic M2 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D018028:Receptors, Neurotensin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D003902:Detergents; D043585:Receptor, Muscarinic M2; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; D000077423:Polidocanol; D011092:Polyethylene Glycols; D013879:Thioredoxins; D004247:DNA; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs; D004072:Digitonin