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- Kronqvist, Nina, et al.
(författare)
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Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display
- 2008
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 278:1, s. 128-136
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Tidskriftsartikel (refereegranskat)abstract
- The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.
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| 2. |
- Lanner, Johanna T., et al.
(författare)
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Knockdown of TRPC3 with siRNA coupled to carbon nanotubes results in decreased insulin-mediated glucose uptake in adult skeletal muscle cells
- 2009
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 23:6, s. 1728-1738
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Tidskriftsartikel (refereegranskat)abstract
- The involvement of Ca2+ in the insulin-mediated signaling cascade, resulting in glucose uptake in skeletal muscle, is uncertain. Here, we test the hypothesis that Ca2+ influx through canonical transient receptor potential 3 (TRPC3) channels modulates insulin-mediated glucose uptake in adult skeletal muscle. Experiments were performed on adult skeletal muscle cells of wild-type (WT) and obese, insulin-resistant ob/ob mice. Application of the diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG) induced a nonselective cation current, which was inhibited by the addition of anti-TRPC3 antibody in the patch pipette and smaller in ob/ob than in WT cells. Knockdown of TRPC3, using a novel technique based on small interfering RNA (siRNA) coupled to functionalized carbon nanotubes, resulted in pronounced (similar to 70%) decreases in OAG-induced Ca2+ influx and insulin-mediated glucose uptake. TRPC3 and the insulin-sensitive glucose transporter 4 (GLUT4) coimmunoprecipitated, and immunofluorescence staining showed that they were colocalized in the proximity of the transverse tubular system, which is the predominant site of insulin-mediated glucose transport in skeletal muscle. In conclusion, our results indicate that TRPC3 interacts functionally and physically with GLUT4, and Ca2+ influx through TRPC3 modulates insulin-mediated glucose uptake. Thus, TRPC3 is a potential target for treatment of insulin-resistant conditions.-Lanner, J. T., Bruton, J. D., Assefaw-Redda, Y., Andronache, Z., Zhang, S.- J., Severa, D., Zhang, Z.- B., Melzer, W., Zhang, S.-L., Katz, A., Westerblad, H. Knockdown of TRPC3 with siRNA coupled to carbon nanotubes results in decreased insulin-mediated glucose uptake in adult skeletal muscle cells. FASEB J. 23, 1728-1738 (2009)
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