Rrectly folded receptor having seven helical transmembrane domains. MaterialDiscussionLarge amounts of OPRM were overproduced as inclusion bodies in RIL cells using the autoinduction method at 37uC when grown for 4 h after induction. The specific degradation into an 18 kD of N-terminal fragment of the receptor was present when expressed with in RP and RIL cells at 37uC. Possibly a flexible intracellular loop exists between the 3rd and 4th transmembrane domain, which was cleaved by a protease from E.coli. RP and RIL cells obviously respond to the over-expression of OPRM 
with cleavage of the membrane protein, whereas C41 and C43 cells direct the protein only into inclusion bodies when buy Tartrazine challenged by expression of OPRM. The effectiveness of the two strains, C41 (DE3) and C43 (DE3), in over-expressing toxic and membrane proteins has been previously demonstrated. The buy [DTrp6]-LH-RH strain C43 (DE3) was derived from C41 (DE3) by selecting for resistance to a different toxic protein [31]. Compared with the other E.coli strains RP and RIL, the C41 and C43 strains were observed to yield more membrane mass per cell mass. This finding may explain one of the reasons whyFigure 3. Solubilisation of OPRM with detergents. A, solubilisation with urea or detergents. B, solubilisation with urea and laurylsarcosine. T -total membrane fraction, S -solubilised membrane fraction, P -pellet after solubilisation. doi:10.1371/journal.pone.0056500.gOPRM from E. coliFigure 4. Purification of OPRM from C43 in Fos-12. A, Purification of OPRM solubilised with Fos-12 by Ni-NTA. T -total membrane fraction, S solubilised membrane fraction, FT -flowthrough. W ?wash fractions (25 mM imidazole), E ?elution fractions (300 mM imidazole). The arrow denotes the monomeric OPRM. B, SEC -purified OPRM after size exclusion chromatography. doi:10.1371/journal.pone.0056500.gOverExpressTM C41 (DE3) and C43 (DE3), have been found to be superior for over-expressing toxic and membrane proteins. The over-expression of OPRM was largely tolerated by C43 with the conditions of 0.4 mM IPTG at 18uC for 8?2 h. Preferential membrane-insertion of OPRM instead of formation of inclusion bodies may be due to the larger mass of membrane in these strains. The membrane insertion of the protein represented first evidence that a correctly folded and stable OPRM was obtained. Interestingly, in contrast to the N-terminally tagged OPRM the expression of C-terminal decahis-tag OPRM in C43 gave only a poor expression level of the protein. This result appears to contradict the conclusion that a hexahistidine tag fused at the amino terminus of opioid receptor decreased expression levels markedly in baculovirus-infected insect cells [32] due to the “positive inside rule” described for E.coli membrane proteins and GPCRs [33]. The so-called positive inside rule states thatFigure 5. Size exclusion chromatography of OPRM in Fos-12. Purification of OPRM was performed in analytical grade Superdex 200 HR 10/30 12926553 size exclusion chromatography. Peak 1 identifies the aggregation of OPRM. The underlined Peak 2 shows the monomeric form of OPRM. doi:10.1371/journal.pone.0056500.gcytoplasmic segments contain more positive charges than extracytoplasmic segments. This is also true for OPRM. It was also previously claimed that due to poor expression of OPRM with a C-terminal his-tag, E.coli may be not a suitable expression system for OPRM [14]. In the light of our results this conclusion appears to be overly generalising. In our case, the expr.Rrectly folded receptor having seven helical transmembrane domains. MaterialDiscussionLarge amounts of OPRM were overproduced as inclusion bodies in RIL cells using the autoinduction method at 37uC when grown for 4 h after induction. The specific degradation into an 18 kD of N-terminal fragment of the receptor was present when expressed with in RP and RIL cells at 37uC. Possibly a flexible intracellular loop exists between the 3rd and 4th transmembrane domain, which was cleaved by a protease from E.coli. RP and RIL cells obviously respond to the over-expression of OPRM with cleavage of the membrane protein, whereas C41 and C43 cells direct the protein only into inclusion bodies when challenged by expression of OPRM. The effectiveness of the two strains, C41 (DE3) and C43 (DE3), in over-expressing toxic and membrane proteins has been previously demonstrated. The strain C43 (DE3) was derived from C41 (DE3) by selecting for resistance to a different toxic protein [31]. Compared with the other E.coli strains RP and RIL, the C41 and C43 strains were observed to yield more membrane mass per cell mass. This finding may explain one of the reasons whyFigure 3. Solubilisation of OPRM with detergents. A, solubilisation with urea or detergents. B, solubilisation with urea and laurylsarcosine. T -total membrane fraction, S -solubilised membrane fraction, P -pellet after solubilisation. doi:10.1371/journal.pone.0056500.gOPRM from E. coliFigure 4. Purification of OPRM from C43 in Fos-12. A, Purification of OPRM solubilised with Fos-12 by Ni-NTA. T -total membrane fraction, S solubilised membrane fraction, FT -flowthrough. W ?wash fractions (25 mM imidazole), E ?elution fractions (300 mM imidazole). The arrow denotes the monomeric OPRM. B, SEC -purified OPRM after size exclusion chromatography. doi:10.1371/journal.pone.0056500.gOverExpressTM C41 (DE3) and C43 (DE3), have been found to be superior for over-expressing toxic and membrane proteins. The over-expression of OPRM was largely tolerated by C43 with the conditions of 0.4 mM IPTG at 18uC for 8?2 h. Preferential membrane-insertion of OPRM instead of formation of inclusion bodies may be due to the larger mass of membrane in these strains. The membrane insertion of the protein represented first evidence that a correctly folded and stable OPRM was obtained. Interestingly, in contrast to the N-terminally tagged OPRM the expression of C-terminal decahis-tag OPRM 
in C43 gave only a poor expression level of the protein. This result appears to contradict the conclusion that a hexahistidine tag fused at the amino terminus of opioid receptor decreased expression levels markedly in baculovirus-infected insect cells [32] due to the “positive inside rule” described for E.coli membrane proteins and GPCRs [33]. The so-called positive inside rule states thatFigure 5. Size exclusion chromatography of OPRM in Fos-12. Purification of OPRM was performed in analytical grade Superdex 200 HR 10/30 12926553 size exclusion chromatography. Peak 1 identifies the aggregation of OPRM. The underlined Peak 2 shows the monomeric form of OPRM. doi:10.1371/journal.pone.0056500.gcytoplasmic segments contain more positive charges than extracytoplasmic segments. This is also true for OPRM. It was also previously claimed that due to poor expression of OPRM with a C-terminal his-tag, E.coli may be not a suitable expression system for OPRM [14]. In the light of our results this conclusion appears to be overly generalising. In our case, the expr.
