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Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy.

Mizrachi D, Chen Y, Liu J, Peng HM, Ke A, Pollack L, Turner RJ, Auchus RJ, DeLisa MP - Nat Commun (2015)

Bottom Line: Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets.However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents.This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic 'shield' that sequesters the IMP from water and promotes its solubilization.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic 'shield' that sequesters the IMP from water and promotes its solubilization.

No MeSH data available.


Related in: MedlinePlus

In vivo solubilization of EmrE using the SIMPLEx strategy.(a) Western blot analysis of soluble (sol), detergent solubilized (det) and insoluble (ins) fractions prepared from E. coli strain BL21(DE3) expressing either EmrE, OspA-EmrE, OspA-ApoAI* or OspA-EmrE-ApoAI* as indicated. Blot was probed with anti-His antibody. Molecular weight (MW) markers are shown on the left. (b) Fluorescence microscopy of BL21(DE3) cells expressing the same constructs in (a) that were each modified with a C-terminal GFP fusion for visualizing protein expression and localization. (c) Ligand-binding activity performed using dimeric, detergent-free OspA-EmrE-ApoAI* or organic-extracted detergent-solubilized EmrE, both of which were purified from BL21(DE3) cells. Assays were performed with EtBr as substrate. Determination of binding constants was based on fluorescence quenching. Data are expressed as the mean of biological quadruplicates and the error, defined as the s.e.m., was <5%.
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f1: In vivo solubilization of EmrE using the SIMPLEx strategy.(a) Western blot analysis of soluble (sol), detergent solubilized (det) and insoluble (ins) fractions prepared from E. coli strain BL21(DE3) expressing either EmrE, OspA-EmrE, OspA-ApoAI* or OspA-EmrE-ApoAI* as indicated. Blot was probed with anti-His antibody. Molecular weight (MW) markers are shown on the left. (b) Fluorescence microscopy of BL21(DE3) cells expressing the same constructs in (a) that were each modified with a C-terminal GFP fusion for visualizing protein expression and localization. (c) Ligand-binding activity performed using dimeric, detergent-free OspA-EmrE-ApoAI* or organic-extracted detergent-solubilized EmrE, both of which were purified from BL21(DE3) cells. Assays were performed with EtBr as substrate. Determination of binding constants was based on fluorescence quenching. Data are expressed as the mean of biological quadruplicates and the error, defined as the s.e.m., was <5%.

Mentions: To solubilize EmrE, a plasmid was created encoding a chimeric protein in which ApoAI* was fused to the C terminus of EmrE. To prevent the secretory pathway in E. coli from inserting EmrE directly into the inner membrane, we introduced a highly soluble ‘decoy' protein from Borrelia burgdorferi, namely outer surface protein A (OspA)23, to the N terminus of the EmrE-ApoAI* chimera. We predicted that the resulting tripartite fusion would partition to the cytoplasm due to the presence of the N-terminal OspA decoy and would give rise to solubilized EmrE due to the proteic shield afforded by ApoAI*. To test this hypothesis, we examined the cellular accumulation of OspA-EmrE-ApoAI* in E. coli cells transformed with pSIMPLEx-EmrE. Western blot analysis of the soluble cytoplasmic fraction recovered from these cells confirmed that the tripartite fusion was a stable, water-soluble protein with hardly any of the fusion protein partitioning to the insoluble fraction (Fig. 1a). Following cell lysis and Ni2+-affinity chromatography in the absence of detergents, we obtained ∼10–15 mg of OspA-EmrE-ApoAI* per litre of culture. Size-exclusion chromatography (SEC) confirmed that the majority of the soluble OspA-EmrE-ApoAI* was dimers and tetramers (Supplementary Fig. 1a), consistent with the earlier observation that the basic functional unit of EmrE is the dimer but may also include a dimer of dimers24. Peaks corresponding to dimers and tetramers were isolated and reapplied to the SEC (Supplementary Fig. 1b). Final yields of both species ranged between 8 and 10 mg l−1 of culture. It is worth noting that the solubility profile of OspA-EmrE-ApoAI* was nearly identical to that of a control fusion, OspA-ApoAI* lacking the IMP, which also accumulated exclusively in the soluble fraction (Fig. 1a). In stark contrast, EmrE expressed alone was detected in the detergent soluble and insoluble fractions only (Fig. 1a). A fusion comprised of OspA and EmrE without the ApoAI* domain accumulated in all three fractions of the lysate (soluble, detergent soluble and insoluble). However, all of the soluble OspA-EmrE was aggregated as confirmed by SEC (Supplementary Fig. 1c). The importance of the decoy was revealed by an EmrE-ApoAI* fusion lacking the OspA decoy, which accumulated in the detergent soluble and insoluble fractions in a manner similar to EmrE expressed alone (Fig. 1a). This insolubility was largely due to EmrE as the ApoAI* domain expressed on its own accumulated in all three fractions of the lysate (Fig. 1a).


Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy.

Mizrachi D, Chen Y, Liu J, Peng HM, Ke A, Pollack L, Turner RJ, Auchus RJ, DeLisa MP - Nat Commun (2015)

In vivo solubilization of EmrE using the SIMPLEx strategy.(a) Western blot analysis of soluble (sol), detergent solubilized (det) and insoluble (ins) fractions prepared from E. coli strain BL21(DE3) expressing either EmrE, OspA-EmrE, OspA-ApoAI* or OspA-EmrE-ApoAI* as indicated. Blot was probed with anti-His antibody. Molecular weight (MW) markers are shown on the left. (b) Fluorescence microscopy of BL21(DE3) cells expressing the same constructs in (a) that were each modified with a C-terminal GFP fusion for visualizing protein expression and localization. (c) Ligand-binding activity performed using dimeric, detergent-free OspA-EmrE-ApoAI* or organic-extracted detergent-solubilized EmrE, both of which were purified from BL21(DE3) cells. Assays were performed with EtBr as substrate. Determination of binding constants was based on fluorescence quenching. Data are expressed as the mean of biological quadruplicates and the error, defined as the s.e.m., was <5%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403311&req=5

f1: In vivo solubilization of EmrE using the SIMPLEx strategy.(a) Western blot analysis of soluble (sol), detergent solubilized (det) and insoluble (ins) fractions prepared from E. coli strain BL21(DE3) expressing either EmrE, OspA-EmrE, OspA-ApoAI* or OspA-EmrE-ApoAI* as indicated. Blot was probed with anti-His antibody. Molecular weight (MW) markers are shown on the left. (b) Fluorescence microscopy of BL21(DE3) cells expressing the same constructs in (a) that were each modified with a C-terminal GFP fusion for visualizing protein expression and localization. (c) Ligand-binding activity performed using dimeric, detergent-free OspA-EmrE-ApoAI* or organic-extracted detergent-solubilized EmrE, both of which were purified from BL21(DE3) cells. Assays were performed with EtBr as substrate. Determination of binding constants was based on fluorescence quenching. Data are expressed as the mean of biological quadruplicates and the error, defined as the s.e.m., was <5%.
Mentions: To solubilize EmrE, a plasmid was created encoding a chimeric protein in which ApoAI* was fused to the C terminus of EmrE. To prevent the secretory pathway in E. coli from inserting EmrE directly into the inner membrane, we introduced a highly soluble ‘decoy' protein from Borrelia burgdorferi, namely outer surface protein A (OspA)23, to the N terminus of the EmrE-ApoAI* chimera. We predicted that the resulting tripartite fusion would partition to the cytoplasm due to the presence of the N-terminal OspA decoy and would give rise to solubilized EmrE due to the proteic shield afforded by ApoAI*. To test this hypothesis, we examined the cellular accumulation of OspA-EmrE-ApoAI* in E. coli cells transformed with pSIMPLEx-EmrE. Western blot analysis of the soluble cytoplasmic fraction recovered from these cells confirmed that the tripartite fusion was a stable, water-soluble protein with hardly any of the fusion protein partitioning to the insoluble fraction (Fig. 1a). Following cell lysis and Ni2+-affinity chromatography in the absence of detergents, we obtained ∼10–15 mg of OspA-EmrE-ApoAI* per litre of culture. Size-exclusion chromatography (SEC) confirmed that the majority of the soluble OspA-EmrE-ApoAI* was dimers and tetramers (Supplementary Fig. 1a), consistent with the earlier observation that the basic functional unit of EmrE is the dimer but may also include a dimer of dimers24. Peaks corresponding to dimers and tetramers were isolated and reapplied to the SEC (Supplementary Fig. 1b). Final yields of both species ranged between 8 and 10 mg l−1 of culture. It is worth noting that the solubility profile of OspA-EmrE-ApoAI* was nearly identical to that of a control fusion, OspA-ApoAI* lacking the IMP, which also accumulated exclusively in the soluble fraction (Fig. 1a). In stark contrast, EmrE expressed alone was detected in the detergent soluble and insoluble fractions only (Fig. 1a). A fusion comprised of OspA and EmrE without the ApoAI* domain accumulated in all three fractions of the lysate (soluble, detergent soluble and insoluble). However, all of the soluble OspA-EmrE was aggregated as confirmed by SEC (Supplementary Fig. 1c). The importance of the decoy was revealed by an EmrE-ApoAI* fusion lacking the OspA decoy, which accumulated in the detergent soluble and insoluble fractions in a manner similar to EmrE expressed alone (Fig. 1a). This insolubility was largely due to EmrE as the ApoAI* domain expressed on its own accumulated in all three fractions of the lysate (Fig. 1a).

Bottom Line: Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets.However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents.This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic 'shield' that sequesters the IMP from water and promotes its solubilization.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic 'shield' that sequesters the IMP from water and promotes its solubilization.

No MeSH data available.


Related in: MedlinePlus