Ession level of the N-terminally his-tagged receptor could be obtained in yields of 0.3?.5 mg/liter of culture, which is the highest yield obtained for GPCRs from E.coli membrane ever reported. The obtained yield of purified OPRM is 0.17 mg/liter of culture, which corresponds to 30?0 of expressed OPRM. Several mild detergents were used for PS 1145 web solubilisation of the receptor, only to find solubilisation efficiency was too low and none of them was able to extract sufficient order 115103-85-0 amounts of receptor except Fos-12, probably due to poor membrane breakage and solubilisation for the target protein. Further investigation of the optimal detergent e.g. Fos-14 may allow increasing the yield:expression ratio even further. The detergent Fos-14 has been reported previously to be efficient for solubilising several other GPCRs [28,34]. The overall result improved both in yield and purity of OPRM, especially for low expression conditions, after removing the periplasmic material before cell lysis. This appears to be due to improved performance of affinity chromatography [35]. The monomeric/dimeric OPRM was separable from the aggregated state of OPRM. Thus, circular dichroism (CD) was further used to assess the state of folding of the receptor: The purified OPRM showed the predicted fraction of a-helical secondary structure as expected for a properly folded receptor, whereas the aggregated material displays reduced helicity. Anyhow, from our results it remains unclear to what extend the formation 25837696 of the aggregated material with lower alpha-helicity is due to thermal or detergent induced instability of the folded protein or a principal difficulty of folding of OPRM in E.coli. We suppose that the membrane-integrated protein is folded. Therefore detergent induced instability appears to be the most likely cause for the appearance of a substantial fraction of protein with reduced secondary structure. We assessed the presence of tertiary structure, respectively functionality, by the ability to bind the agonist EM-1. A KD ofOPRM from E. coliFigure 6. Mass spectrometry of OPRM. Sequence coverage of trypsin digested peptide fragments identified. MS/MS spectrum of an identified peptide fragment EFCIPTSSNIEQQNSTR and OPRM sequence with identified fragments in red. doi:10.1371/journal.pone.0056500.gOPRM for EM-1 (61618 nM) was determined by Surface Plasma Resonance, which is 
comparable to the value published for receptor from HEK293 cells (29.962.9 nM) [36], if methodological differences are taken into account. Yet, agonist affinity was decreased by presumably two orders of magnitude as compared to the value measured from mammalian cells for EM-1 (360 pM) [37]. It was presumed previously that the difference between the affinity for EM-1 (29.9 nM) and that first reported value (0.36 nM) is due to the use of different receptor preparations and radioligands [36]. The effect of mammalian lipids could also explain the substantial difference [38]. Finally, our results on a human membrane protein, respectively GPCR, that has been previously proven to be very difficult toexpress, provide further evidence that a moderate expression level and a slow expression rate at low temperature should be targeted in E.coli. The easy scale up and speed of expression in E.coli compensates for the moderate yield, which is still sufficient to allow performing even crystallization experiments.Materials and Methods MaterialsE. coli cell strains CodonPlus RP and CodonPlus RIL were purchased from Strata.Ession level of the N-terminally his-tagged receptor could be obtained in yields of 0.3?.5 mg/liter of culture, which is the highest yield obtained for GPCRs from E.coli membrane ever reported. The obtained yield of purified OPRM is 0.17 mg/liter of culture, which corresponds to 30?0 of expressed OPRM. Several mild detergents were used for solubilisation of the receptor, only to find solubilisation efficiency was too low and none of them was able to extract sufficient amounts of receptor except Fos-12, probably due to poor membrane breakage and solubilisation for the target protein. Further investigation of the optimal detergent e.g. Fos-14 may allow increasing the yield:expression ratio even further. The detergent Fos-14 has been reported previously to be efficient for solubilising several other GPCRs [28,34]. The overall result improved both in yield and purity of OPRM, especially for low expression conditions, after removing the periplasmic material before cell lysis. This appears to be due to improved performance of affinity chromatography [35]. The monomeric/dimeric OPRM was separable from the aggregated state of OPRM. Thus, circular dichroism (CD) was further used to assess the state of folding of the receptor: The purified OPRM showed the predicted fraction of a-helical secondary structure as expected for a properly folded receptor, whereas the aggregated material displays reduced helicity. Anyhow, from our results it remains unclear to what extend the formation 25837696 of the aggregated material with lower alpha-helicity is due to thermal or 
detergent induced instability of the folded protein or a principal difficulty of folding of OPRM in E.coli. We suppose that the membrane-integrated protein is folded. Therefore detergent induced instability appears to be the most likely cause for the appearance of a substantial fraction of protein with reduced secondary structure. We assessed the presence of tertiary structure, respectively functionality, by the ability to bind the agonist EM-1. A KD ofOPRM from E. coliFigure 6. Mass spectrometry of OPRM. Sequence coverage of trypsin digested peptide fragments identified. MS/MS spectrum of an identified peptide fragment EFCIPTSSNIEQQNSTR and OPRM sequence with identified fragments in red. doi:10.1371/journal.pone.0056500.gOPRM for EM-1 (61618 nM) was determined by Surface Plasma Resonance, which is comparable to the value published for receptor from HEK293 cells (29.962.9 nM) [36], if methodological differences are taken into account. Yet, agonist affinity was decreased by presumably two orders of magnitude as compared to the value measured from mammalian cells for EM-1 (360 pM) [37]. It was presumed previously that the difference between the affinity for EM-1 (29.9 nM) and that first reported value (0.36 nM) is due to the use of different receptor preparations and radioligands [36]. The effect of mammalian lipids could also explain the substantial difference [38]. Finally, our results on a human membrane protein, respectively GPCR, that has been previously proven to be very difficult toexpress, provide further evidence that a moderate expression level and a slow expression rate at low temperature should be targeted in E.coli. The easy scale up and speed of expression in E.coli compensates for the moderate yield, which is still sufficient to allow performing even crystallization experiments.Materials and Methods MaterialsE. coli cell strains CodonPlus RP and CodonPlus RIL were purchased from Strata.
