Mistic and TarCF as fusion protein partners for functional expression of the cannabinoid receptor 2 in Escherichia coli

Abstract

G protein coupled receptors (GPCRs) are key players in signal recognition and cellular communication making them important therapeutic targets. Large-scale production of these membrane proteins in their native form is crucial for understanding their mechanism of action and target-based drug design. Here we report the overexpression system for a GPCR, the cannabinoid receptor subtype 2 (CB2), in Escherichia coli C43(DE3) facilitated by two fusion partners: Mistic, an integral membrane protein expression enhancer at the N-terminal, and TarCF, a C-terminal fragment of the bacterial chemosensory transducer Tar at the C-terminal of the CB2 open reading frame region. Multiple histidine tags were added on both ends of the fusion protein to facilitate purification. Using individual and combined fusion partners, we found that CB2 fusion protein expression was maximized only when both partners were used. Variable growth and induction conditions were conducted to determine and optimize protein expression. More importantly, this fusion protein Mistic-CB2-TarCF can localize into the E. coli membrane and exhibit functional binding activities with known CB2 ligands including CP55,940, WIN55,212-2 and SR144528. These results indicate that this novel expression and purification system provides us with a promising strategy for the preparation of biologically active GPCRs, as well as general application for the preparation of membrane-bound proteins using the two new fusion partners described.

Advances in Methods to Characterize Ligand-Induced Ionic Lock and Rotamer Toggle Molecular Switch in G Protein-Coupled Receptors

“…Although Escherichia coli has obvious advantages of fast and low-cost production of GPCR and/or GPCR fragments, receptor may not be translocated to the membrane and hence render nonfunctional. Substantial success has been achieved for the production of the GPCR fragments in the E. coli (Chowdhury et al, 2012; Xie, Zheng, & Zhao, 2004; Zhang & Xie, 2008; Zheng et al, 2005). Expression and posttranslational modification of recombinant GPCR is similar to that of mammalian cells, and GPCR produced are almost identical in insect and mammalian cells except for some minor differences.…”

N-Terminal Fusion Tags for Effective Production of G-Protein-Coupled Receptors in Bacterial Cell-Free Systems

“…For further optimization of the synthesis for the model GPCRs, we tested four N-terminal fusion tags. Two of those, the T7-tag (11 a.a.) and TRX protein (11.8 kDa), have previously been used in cell-free production of GPCRs [ 14 , 16 , 17 ], whereas the Mistic protein (12.8 kDa) was used for GPCR production in E. coli [ 31 , 32 ]. In addition, we tested the sequence encoding the N-terminal fragment (16 a.a.) of ribonuclease A (N-terminal fragment of S-peptide, S-tag), which is used to detect and purify recombinant proteins via affinity chromatography [ 33 ], but has never been used as an N-terminal fusion tag for the production of recombinant MPs.…”

Large-scale production and protein engineering of G protein-coupled receptors for structural studies

“…Even higher expression levels were obtained if, in addition to MBP as the N-terminal fusion partner, TrxA was added at the C-terminus ( Tucker and Grisshammer, 1996 ; Furukawa and Haga, 2000 ; Yeliseev et al, 2007 ). More recently, combination of N-terminally attached Mistic and C-terminally attached TarCF gave functional expression of human CB 2 receptor in E. coli ( Chowdhury et al, 2012 ).…”