Ut 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 1655472 or autoinduction) were varied to optimize the expression level. At high temperature (37uC), the N-terminal his-tagged OPRM was found to be produced both in inclusion bodies and in membrane-inserted form (BIRB 796 web 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. 16402044 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 Dinaciclib 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. Therefore a variety of detergents were used to extract OPRM from E.coli membrane and as controls: Zwitterionic detergents (1 (w/v) LDAO, 1 (w/v) Fos-12), nonionic detergents (1 (w/v) DDM, 1 (w/v) Cy6) and anionic detergent (1 (w/v) SDS, 0.8 (w/v) laurylsarcosine with/without 6 M urea). The detergents for the isolation of folded protein were chosen to cover the typical range of micelle aggregation numbers (10?33) and a reduced range of hydrophile-lipophile balances (HLB: 5.3 to 14.2) [30]. The more hydrophilic detergents with HLB.14.2 were excluded because complete solubilisation of the target protein was aimed for. Urea without detergent showed very poor solubilisation efficiency. The receptor remained in the pellet upon solubilisation, indicati.Ut 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 1655472 or autoinduction) were varied to optimize the expression level. At high temperature (37uC), the N-terminal his-tagged OPRM was found to be 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. 16402044 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. Therefore a variety of detergents were used to extract OPRM from E.coli membrane and as controls: Zwitterionic detergents (1 (w/v) LDAO, 1 (w/v) Fos-12), nonionic detergents (1 (w/v) DDM, 1 (w/v) Cy6) and anionic detergent (1 (w/v) SDS, 0.8 (w/v) laurylsarcosine with/without 6 M urea). The detergents for the isolation of folded protein were chosen to cover the typical range of micelle aggregation numbers (10?33) and a reduced range of hydrophile-lipophile balances (HLB: 5.3 to 14.2) [30]. The more hydrophilic detergents with HLB.14.2 were excluded because complete solubilisation of the target protein was aimed for. Urea without detergent showed very poor solubilisation efficiency. The receptor remained in the pellet upon solubilisation, indicati.
