E coli Expression

Because of the simple and rapid procedure for expression and scale-up of recombinant protein production, GPCRs have been evaluated in E. coli. As bacterial cells lack many of the post-translational modification mechanisms in mammalian cells such as glycosylation, the functionality of GPCRs expressed in bacteria is of certain concern. Two alternative approaches involve expressing the recombinant receptors in the plasma membrane or in bacterial inclusion bodies. Both methods have stringent requirements for achieving success.

The insertion of foreign and especially mammalian receptors in bacterial membranes appears toxic to host cells and results in slow growth rates of bacteria, which significantly reduces yields. To overcome these problems, deletions have been introduced in the GPCR sequences and fusion constructs engineered.5 Expression in bacterial inclusion bodies generally produces relatively high yields, but the requirement for refolding of receptors prior to purification and crystallography has hampered progress. Although some success has been achieved for the refolding as described below, it still represents a major bottleneck in structural biology.

One of the most successful GPCR expression studies in E. coli membranes was obtained for the rat neurotensin receptor (NTR).5 The N-terminus of the NTR was deleted and replaced by the MBP and a C-terminal bio-tag (in vivo biotinylated) that interacts with avidin. The MBP-NTR fusion protein was expressed in a functional form in milligram quantities and was subjected to a two-step purification procedure consisting of a monomeric avidin column followed by a neurotensin affinity column. Purification of the rat MBP-NTR from E. coli produced milligram amounts of a functional receptor that was reconstituted in lipid vesicles and applied for solid state NMR (described in detail in Chapter 13) bound to a high-affinity neurotensin peptide agonist.14

In another study, the human adenosine A2a receptor (hA2aR) was overexpressed as a fusion protein with MBP in the inner membranes at levels of 10 to 20 nmol receptor/L culture.15 A truncation at Ala316 at the C-terminus made the receptor protease resistant, although still functional, which allowed establishment of a three-

step ligand affinity purification procedure of fully functional hA2aR. Additionally, when the human serotonin 5-HT1A receptor was fused to the signal sequence of the maltose binding protein MalE, active receptor expression was obtained in the inner bacterial membrane measured by ligand binding.16

Expression in bacterial inclusion bodies has been studied for both truncated and full-length GPCRs. The N-terminal extracellular domain of the human parathyroid hormone receptor 1 (PTHR1) was expressed at high levels in E. coli inclusion bodies.17 A stable, soluble monomeric receptor was obtained after oxidative refolding for which ligand binding was demonstrated by surface plasmon resonance spectroscopy and isothermal titration calorimetry. Circular dichroism indicated that the refolded material contained secondary structures of approximately 25% a-helices and 23% p-sheets.

The extracellular region of the human glucagon-like peptide 1 receptor (GLP-1R) was expressed in bacterial inclusion bodies, for which renaturation was obtained from guanidinium-solubilized material.18 After ion exchange chromatography and gel filtration, the activity of GLP-1R was measured by cross-linking, surface plasmon resonance, and isothermal titration calorimetry. The extracellular domain of the GLP-1R was purified and refolded and monitored for binding activity. Interestingly, high affinity binding was observed for the peptide antagonist 125I-exendin-4 (9-39), whereas 125I-labelled GLP-1 showed little binding, suggesting that the endogenous agonist GLP-1 might require the presence of extracellular loops and transmembrane regions.19 In another study, 11 GPCRs were expressed with N-terminal GST- and C-terminal His tags in E. coli inclusion bodies at levels varying from 0.1 to 10% of the total cellular protein.20 Refolding protocol technologies have been established for several GPCRs although the yields of activated receptors are relatively poor.21

The expression of the human leukotriene B4 (LTB4) receptor BLT1 in bacterial inclusion bodies was optimized by replacing the codons with the lowest abundance in the E. coli genome with the most frequent codons.22 This procedure led to high expression levels but the receptor was insoluble and misfolded in bacterial inclusion bodies. The C-terminally tagged BLT1 receptor was purified by metal affinity chromatography in urea and refolding was obtained by removing urea from the Ni-NTA-bound material in the presence of the lauryldimethylamino oxide (LDAO) detergent. Circular dichroism analysis suggested a structure with approximately 50% a-helices. Further structural studies indicated that both the LTB4 agonist and the BLT1 receptor undergo conformational changes during their interaction.

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