SIMPLEx, an Amphipathic Protein Fusion Strategy for Making Water-soluble Integral Membrane Proteins
- 详细技术说明
- This invention describesa new method called SIMPLEx (Solubilization of IMPs with high Levelsof Expression), an in vivosolubilization method of Integral Membrane Proteins (IMPs) in functionallyrelevant conformations without the need for detergents or mutations to the IMPitself.
- *Abstract
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Integral membraneproteins (IMPs) play crucial roles in cells and represent attractivepharmacological targets. Progress to understandtheir structure and function has lagged due to their hydrophobic nature. Currentmethods of solubilization involve harsh detergents resulting to extracted IMPswith poor stability and low yields of active proteins. IMPs solubilized by thesemethods can only be investigated in vitro.
DeLisa group has developed an in vivo method called SIMPLEx that exploitsthe affinity of IMPs for hydrophobic surfaces. The team has identified a suitable fusionpartner to IMPs, apolipoprotein A-I (ApoAI), which exhibits remarkablestructural flexibility and serves as an amphipathic proteic “shield” thatsequesters the IMP from water and promotes its solubilization. The construct of the SIMPLEx method involves:
- An amphipathic protein such as a truncated ApoAI;
- An IMP target (polytopic α-helical IMPs orpolytopic β-barrel); and,
- A decoy protein such as OspA or E. coli maltose-binding protein lackingits native export signal peptide (cMBP).
Chimeras resulting from the SIMPLExmethod yield appreciable amounts of IMPs in the cytoplasm and render them highlysoluble in the absence of detergents. Those solubilized IMPs are stable, preservetheir functionality, and are amenable to structural characterization.
Proof of concept: The team targeted the bacterial integralmembrane enzyme DsbB that generates disulfide bond in E. coli (figure 1a). Using the SIMPLEx method, they convertedmembrane-bound DsbB into a water-soluble biocatalyst that could be readilyexpressed in the cytoplasm (Figure 1b). The solubilized DsbB in presence of cDsbAwas able to oxidize the cytoplasmic variant of alkaline phosphatase, cPhoA, andto catalyze disulfide-bond formation critical for both stability andphosphatase activity (figure 2).
PotentialApplications
- Production of water-soluble membrane proteins
- Tool to characterize function and structure of membraneproteins; particularly proteins used in biological pathways such asglycosylation.
Advantages
- Efficientin stabilizing and folding membrane proteins
- Nodetergents or lipid reconstitutions required
- Enzymaticactivity of proteins maintained
- Appreciableoverexpression yield
- Generalizablemethod to either polytopic α-helical or polytopic β-barrel IMPs
- Allowsfor expression of membrane proteins in the cytoplasm or other cellularcompartments
- Mightallow for cell-free use of membrane proteins.
- *Licensing
- Phillip Owh607-254-4508po62@cornell.edu
- 其他
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- Patentfiled in the U.S. US20170275343
- DarioMizrachi & al. (2015). Making water-soluble integral membrane proteins in vivo using an amphipathic proteinfusion strategy. Nature Communications 6:6826.DOI:10.1038/ncomms7826;
- DarioMizrachi & al. (2017). A water-soluble DsbB variant that catalyzesdisulfide-bond formation in vivo. Nature Chemical Biology 13, 1022–1028. doi:10.1038/nchembio.2409;
- 'Shield' gives tricky proteins a newidentity – By Anne Ju, April9, 2015;
- ‘Changing the identity of cellularenzyme spawns new pathway’By Tom Fleischman June 20, 2017.
- 国家/地区
- 美国
