Restoring full biological activity to the isolated ectodomain of an integral membrane protein

Biochemistry
Emily K Waters, J H Morrissey

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 co...Continue Reading

References

Oct 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·D RepkeY Nemerson
Nov 1, 1988·Thrombosis Research·J H MorrisseyT S Edgington
Mar 27, 1998·FEBS Letters·D T Jones
May 6, 1998·Protein Science : a Publication of the Protein Society·E Wallin, G von Heijne
Jul 8, 1998·Proceedings of the National Academy of Sciences of the United States of America·S A Darst
Sep 6, 2003·Journal of Immunological Methods·Jay T Groves, Michael L Dustin
Jun 29, 2004·Journal of Thrombosis and Haemostasis : JTH·S A Smith, J H Morrissey
Sep 24, 2004·Current Medicinal Chemistry·Robert A LazarusDaniel Kirchhofer
Nov 3, 2004·Biochimica Et Biophysica Acta·Annela M SeddonPaula J Booth
Jan 22, 2005·Annual Review of Medicine·Lori-Ann Linkins, Jeffrey I Weitz

❮ Previous
Next ❯

Citations

May 13, 2011·The Journal of Biological Chemistry·Narjes TavoosiJames H Morrissey
Jun 15, 2007·Journal of Thrombosis and Haemostasis : JTH·D M Monroe, N S Key
Aug 22, 2012·Journal of Thrombosis and Haemostasis : JTH·N J MutchJ H Morrissey
Jul 25, 2013·Biochemistry·Narjes TavoosiJames H Morrissey
Jan 29, 2008·Chemical Biology & Drug Design·Anthony L ShroutRobert M Weis
Jan 4, 2007·The Journal of Biological Chemistry·Andrew W ShawJames H Morrissey
Oct 4, 2008·Journal of Biomolecular Screening·Edward A EspositoRobert M Weis
Sep 5, 2020·Journal of Thrombosis and Haemostasis : JTH·Fabienne Birkle, James H Morrissey
Jul 15, 2020·The Journal of Clinical Investigation·Sol SchulmanBruce Furie
Jan 30, 2009·Langmuir : the ACS Journal of Surfaces and Colloids·Rainer NehringWolfgang Meier

❮ Previous
Next ❯

Related Concepts

Related Feeds

Blood Clotting Disorders

Thrombophilia includes conditions with increased tendency for excessive blood clotting. Blood clotting occurs when the body has insufficient amounts of specialized proteins that make blood clot and stop bleeding. Here is the latest research on blood clotting disorders.