Improved yield of a ligand-binding GPCR expressed in E. coli for structural studies.
The G-protein coupled receptor (GPCR), rat brain neurotensin receptor type I (NTS1) is one of a small number of GPCRs that have been successfully expressed in Escherichia coli as a functional, ligand-binding receptor, but yields of purified receptor are still low for comprehensive structural studies. Here, several approaches have been examined to optimize the yields of active, ligand-binding receptor. Optimisation of E. coli strain and induction protocol yielded a significant improvement in expression of active receptor. Expression of the receptor in BL21(DE3) cells, in combination with autoinduction improved expression 10-fold when compared with previously reported expression protocols using IPTG-mediated induction in DH5alpha cells. Optimization of the purification protocol revealed that supplementation of buffers with phospholipids enhanced recovery of active receptor. The methods examined are potentially applicable to other GPCRs expressed in E. coli.
Attrill
Helen
H
Biochemistry Department, University of Oxford, Oxford, UK.
Harding
Peter J
PJ
Smith
Eleanor
E
Ross
Simon
S
Watts
Anthony
A
D000595:Amino Acid Sequence; D000818:Animals; D002021:Buffers; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008969:Molecular Sequence Data; D009496:Neurotensin / Q000302:isolation & purification / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018028:Receptors, Neurotensin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013696:Temperature
D002021:Buffers; D008024:Ligands; D010743:Phospholipids; D043562:Receptors, G-Protein-Coupled; D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; C114036:neurotensin type 1 receptor; D009496:Neurotensin