Abstract：
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Waters EK,Morrissey JHdoi
10.1021/bi052600msubject
Has Abstractpub_date
2006-03-21 00:00:00pages
3769-74issue
11eissn
0006-2960issn
1520-4995journal_volume
45pub_type
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