title: Efficient production and purification of recombinant murine kindlin-3 from insect cells for biophysical studies.
abstract: Kindlins are essential coactivators, with talin, of the cell surface receptors integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin β-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3's role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford; luke@strubi.ox.ac.uk.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Yates
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford; gilbert@strubi.ox.ac.uk.
authors.forename: Robert J C
authors.initials: RJ
authors.lastname: Gilbert
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001703:Biophysics / Q000379:methods; D003598:Cytoskeletal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D061987:Sf9 Cells; D018411:Spodoptera / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D003598:Cytoskeletal Proteins; D011994:Recombinant Proteins; C527818:kindlin-3 protein, mouse