Engineering the lac permease for purification and crystallization.
The lactose permease is being used as a model system for the rational redesign of a membrane protein with the goal of increasing the likelihood of crystallization. Various modifications to the protein have been added for the purposes of purification, stability, and potential for crystallization. The addition of six consecutive histidines at the C-terminus of the protein allows for rapid purification by nickel-chelate chromatography, and the insertion of an entire protein domain into one of the inner cytoplasmic loops of the permease gives the resulting protein a larger hydrophilic surface area. The increase in polar surface area makes the fusion protein easier to handle and more likely to crystallize. In particular, the introduction of cytochrome b562 of E. coli into the central hydrophilic domain the lac permease results in a fusion protein with the transport activity of the permease and the visible absorbance spectrum of the cytochrome. The "red permease" is very easy to monitor through the steps of expression, purification, concentration, and crystallization.
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Ontario Cancer Institute, University of Toronto, Canada.
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D001360:Avidin; D003460:Crystallization; D003573:Cytochrome b Group / Q000235:genetics / Q000302:isolation & purification; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000302:isolation & purification*; D009004:Monosaccharide Transport Proteins; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009942:Organometallic Compounds; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D013053:Spectrophotometry; D027981:Symporters
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D009942:Organometallic Compounds; D011993:Recombinant Fusion Proteins; D027981:Symporters; D001360:Avidin; C088321:nickel nitrilotriacetic acid; C017850:cytochrome b562, E coli; C021499:lactose permease; D009571:Nitrilotriacetic Acid