Ids problems with posttranslational modifications and GPCR heterooligomerization with GPCRs of

Ids problems with posttranslational modifications and GPCR heterooligomerization with GPCRs of the host cells [25]. However, overexpression of membrane proteins in membrane-integrated form in E.coli is usually toxic to the organism and thus leads to reduction in yields [26], presumably due to the limitation of the E.coli membrane space and different membrane translocation system. It has been reported that several functional GPCRs were successfully expressed via E.coli [12,14,23,27] or E.coli cell-free system 1676428 [11,28,29]. Here, we investigated the possibility to obtain by heterologous expression in E.coli functional human mu-opioid receptor, which is modified only by a removable his-tag to facilitate enrichment and identification upon purification, but does not contain any stabilizing modifications like insertion of T4 lysozyme [10] that may affect the expected structural changes of the receptor when performing the signaling function.OPRM from E. coliResults Expression of a Membrane-inserted OPRM in E.coliVarious E.coli strains (RP, RIL, C41, and C43) were screened for expression of the target protein. The parameters temperature (18uC and 37uC), induction time, expression medium (DYT and TB) and induction method (0.2?.8 mM IPTG or autoinduction) were varied to (��)-Imazamox price optimize the expression level. At high temperature (37uC), the N-terminal his-tagged OPRM was found to be 25837696 produced both in inclusion bodies and in membrane-inserted form (Figure 1A): for C41 cells only a low expression level was observed, most of the target Tetracosactide protein was found in the inclusion bodies. For other cells at higher expression levels OPRM was increasingly found in form of inclusion bodies or even degraded as seen for the case of expression in RIL cells, where 30?50 of OPRM was degraded into a large N-terminal fragment (ca. 18 k Da). Upon induction with IPTG at 37uC severe foam formation with loss of cell density was observed. Typically the culture decayed within 3 hours after induction. Thus the expression of the OPRM was found to be toxic. Very slow growth of the culture was observed for induction at 18uC. These results indicated a proper harvesting time and induction period should be optimized even for expression at 18uC. Extended induction time (.12 h) led to low cell density (OD600,2), whereas a proper induction time of less than 10 h was optimal to maximize cell yield (Final OD600 = 2?, cell pellet .8 g/l) in all cases. With the richer medium TB more cells could be harvested (Figure 2). The optimized IPTG concentration (0.4 mM) was found to effectively induce the expression of OPRM, while increasing IPTG concentration led to degradation of the protein or to the formation of inclusion bodies. With the conditions of 0.4 mM IPTG at 18uC for 8?2 h in C43 almost no inclusion bodies were produced within C43. OPRM was obtained in the membrane fraction (Figure 1B). The optimal expression level of OPRM was determined to be 0.3?.5 mg/liter of culture by complete solubilisation of the protein in the membrane fraction under denaturating conditions with 6 M urea and 0.8 laurylsarcosine (Figure 3B) and subsequent western blot. Remarkably, no appreciable expression of OPRM with a Cterminal his-tag was observed under any of the tested conditions (data not shown).OPRM SolubilisationSolubilisation of membrane protein from the membrane is one of the crucial steps of purification, which is routinely achieved by optimizing the detergent to minimize denaturation during solubilisation. The.Ids problems with posttranslational modifications and GPCR heterooligomerization with GPCRs of the host cells [25]. However, overexpression of membrane proteins in membrane-integrated form in E.coli is usually toxic to the organism and thus leads to reduction in yields [26], presumably due to the limitation of the E.coli membrane space and different membrane translocation system. It has been reported that several functional GPCRs were successfully expressed via E.coli [12,14,23,27] or E.coli cell-free system 1676428 [11,28,29]. Here, we investigated the possibility to obtain by heterologous expression in E.coli functional human mu-opioid receptor, which is modified only by a removable his-tag to facilitate enrichment and identification upon purification, but does not contain any stabilizing modifications like insertion of T4 lysozyme [10] that may affect the expected structural changes of the receptor when performing the signaling function.OPRM from E. coliResults Expression of a Membrane-inserted OPRM in E.coliVarious E.coli strains (RP, RIL, C41, and C43) were screened for expression of the target protein. The parameters temperature (18uC and 37uC), induction time, expression medium (DYT and TB) and induction method (0.2?.8 mM IPTG or autoinduction) were varied to optimize the expression level. At high temperature (37uC), the N-terminal his-tagged OPRM was found to be 25837696 produced both in inclusion bodies and in membrane-inserted form (Figure 1A): for C41 cells only a low expression level was observed, most of the target protein was found in the inclusion bodies. For other cells at higher expression levels OPRM was increasingly found in form of inclusion bodies or even degraded as seen for the case of expression in RIL cells, where 30?50 of OPRM was degraded into a large N-terminal fragment (ca. 18 k Da). Upon induction with IPTG at 37uC severe foam formation with loss of cell density was observed. Typically the culture decayed within 3 hours after induction. Thus the expression of the OPRM was found to be toxic. Very slow growth of the culture was observed for induction at 18uC. These results indicated a proper harvesting time and induction period should be optimized even for expression at 18uC. Extended induction time (.12 h) led to low cell density (OD600,2), whereas a proper induction time of less than 10 h was optimal to maximize cell yield (Final OD600 = 2?, cell pellet .8 g/l) in all cases. With the richer medium TB more cells could be harvested (Figure 2). The optimized IPTG concentration (0.4 mM) was found to effectively induce the expression of OPRM, while increasing IPTG concentration led to degradation of the protein or to the formation of inclusion bodies. With the conditions of 0.4 mM IPTG at 18uC for 8?2 h in C43 almost no inclusion bodies were produced within C43. OPRM was obtained in the membrane fraction (Figure 1B). The optimal expression level of OPRM was determined to be 0.3?.5 mg/liter of culture by complete solubilisation of the protein in the membrane fraction under denaturating conditions with 6 M urea and 0.8 laurylsarcosine (Figure 3B) and subsequent western blot. Remarkably, no appreciable expression of OPRM with a Cterminal his-tag was observed under any of the tested conditions (data not shown).OPRM SolubilisationSolubilisation of membrane protein from the membrane is one of the crucial steps of purification, which is routinely achieved by optimizing the detergent to minimize denaturation during solubilisation. The.