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.
Highlights
Proteins of the kindlin family are a crucial component of focal adhesion assembly, and essential for complex life
The viral DNA is rescued by recombination with a transfer vector that in this case includes the kindlin-3 gene (FERMT3) and results in the FERMT3 gene replacing the virus very late gene, which is highly expressed but redundant, resulting in a recombinant virus that expresses mouse kindlin-3 as part of the virus life cycle[28]. We identified this method for the production of kindlin-3 after attempts to express and purify it in other expression hosts proved prohibitively difficult and due to the versatility of the pOPIN vector suite, which we used for cloning and that can deployed in many expression hosts[29]
The generation of recombinant baculovirus is achieved by cotransfecting Sf9 cells with mouse kindlin-3 (FERMT3)-containing plasmid together with an engineered linearized This method of baculovirus generation bacmid (BAC10:KO1629) and results in 100% recombinant harvesting the newly formed virions after 5-7 days, as viruses and essentially dispenses the need for plaque shown in Figure 1A. purification[28,30]
Summary
Proteins of the kindlin family are a crucial component of focal adhesion assembly, and essential for complex life. We here describe methods for the production of milligram quantities of recombinant mouse kindlin-3 in insect cell culture, suitable for extensive structural studies and biochemical analysis. In this protocol we make use of an engineered knockout bacmid (BAC10KO:1629) that is, alone, unable to produce viable virions[28]. The plasmid is engineered so that the FERMT3 gene (kindlin-3) is under the control of the p10 baculoviral promoter and the vector contains 5′ UTR/ORF603 and ORF 1629 and encodes a C-terminal His6-tag for downstream purification[29]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
