Sly to be efficient for solubilising several other GPCRs [28,34]. The overall

Sly 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 ASP2215 biological activity results it remains unclear to what extend the formation 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 1317923 cell strains CodonPlus RP and CodonPlus RIL were purchased from Stratagene. OverExpressTM C41 (DE3) and C43 (DE3) were purchased from Lucigen. DNA encoding the humanopioid receptor was provided by Qiagen (Germany). Ni-NTA was purchased from Qiagen (Germany). Superdex 200 (16/60) and analytical grade Superdex 200 HR 10/30 size exclusion chromatography were from GE Healthcare. All other chemicals were from either Sigma-Aldrich or Fluka. Fos-12 was purchased from Anatrace (Maumee, OH) and other detergents were purchased from GLYCON (Germany). Buffer A: 20 mM Tris Cl, 150 mM NaCl, 10 GLPG0634 Glycerol, pH 8. Solubilisation buffer: 20 mM Tris?HCl, 300 mM NaCl, 10 Glycerol, pH 8, 1 Fos-12, 5 mM imidazole. Wash buffer: 20 mM Tris Cl, 300 mM NaCl, 10 Glycerol, pH 8, 0.1 Fos-12, 25 mM imidazole. Elution buffer: 20 mM Tris Cl, 300.Sly 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 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 1317923 cell strains CodonPlus RP and CodonPlus RIL were purchased from Stratagene. OverExpressTM C41 (DE3) and C43 (DE3) were purchased from Lucigen. DNA encoding the humanopioid receptor was provided by Qiagen (Germany). Ni-NTA was purchased from Qiagen (Germany). Superdex 200 (16/60) and analytical grade Superdex 200 HR 10/30 size exclusion chromatography were from GE Healthcare. All other chemicals were from either Sigma-Aldrich or Fluka. Fos-12 was purchased from Anatrace (Maumee, OH) and other detergents were purchased from GLYCON (Germany). Buffer A: 20 mM Tris Cl, 150 mM NaCl, 10 Glycerol, pH 8. Solubilisation buffer: 20 mM Tris?HCl, 300 mM NaCl, 10 Glycerol, pH 8, 1 Fos-12, 5 mM imidazole. Wash buffer: 20 mM Tris Cl, 300 mM NaCl, 10 Glycerol, pH 8, 0.1 Fos-12, 25 mM imidazole. Elution buffer: 20 mM Tris Cl, 300.