Restoring full biological activity to the isolated ectodomain of an integral membrane protein.
Integral membrane proteins, which include many cellular effector proteins and drug targets, can be difficult to produce, purify, and manipulate. Although the isolated ectodomains of many membrane proteins can be expressed as water soluble proteins, biological activity is frequently lost when these proteins are released from the membrane surface. An example is tissue factor, the integral membrane protein that triggers the blood clotting cascade and for which membrane anchoring is essential. Its isolated ectodomain (soluble tissue factor) can be expressed with high yield in bacteria but is orders of magnitude less active than the intact, membrane-anchored protein. We now report full restoration of biological activity to the isolated tissue factor ectodomain via the engineering of a hexahistidine tag onto its C-terminus and its use in combination with membrane bilayers containing nickel-chelating lipids. When soluble tissue factor was tethered to the membrane surface via such metal-chelating lipids, it bound factor VIIa with the same high affinity as wild-type tissue factor, and the resulting factor VIIa-tissue factor complexes supported factor X activation and factor VII autoactivation with essentially wild-type enzyme kinetic constants. Furthermore, when such bilayers were immobilized onto solid supports, they efficiently captured histidine-tagged soluble tissue factor directly from crude culture supernatants, with full biological activity, obviating the need for purification or laborious membrane reconstitution procedures. This strategy is rapid, efficient, scalable, and automatable and should be applicable to other integral membrane proteins, especially those with a single transmembrane domain. Applications include high-throughput screening of mutants or drugs, flow reactors, clinical assays, and point-of-care instrumentation.
Waters
Emily K
EK
Department of Biochemistry, College of Medicine, University of Illinois, Urbana, Illinois 61801, USA.
Morrissey
James H
JH
D000818:Animals; D001921:Brain / Q000378:metabolism; D002614:Chelating Agents / Q000378:metabolism; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D015942:Factor VIIa / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013925:Thromboplastin / Q000378:metabolism / Q000494:pharmacology
D002614:Chelating Agents; C471213:His-His-His-His-His-His; D008081:Liposomes; D008565:Membrane Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa