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Immunology and Infection

رصد الجمعية ليفرز البكتيرية الفوعة عن طريق عامل يشابك بمواقع محددة

Published: December 17, 2013 doi: 10.3791/51217

Materials

Name Company Catalog Number Comments
QuikChange II Site-Directed Mutagenesis Kit Agilent 200521
BPA (H-p-Bz-Phe-OH) Bachem F-2800
TRAN35S-LABEL, Metabolic Labeling Reagent (35S-L-methionine and 35S-L-cysteine, >1,000 Ci/mmol) MP Biomedicals 51006
Spectroline SB-100P Super-High-Intensity UV Lamp, 365 nm Spectronics Corporation SB-110P
Spectroline Replacement Bulb 100S Spectronics Corporation 11-992-15
Falcon Tissue Culture Plate, 6-well Becton Dickinson Labware 353046
Disposable 125 ml Erlenmeyer flask Corning 430421
Innova 3100 shaking water bath New Brunswick Scientific n.a.

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References

  1. Wang, L., Xie, J., Schultz, P. G. Expanding the genetic code. Annu. Rev. Biophys. Biomol. Struct. 35, 225-249 (2006).
  2. von Mering, C., et al. Comparative assessment of large-scale data sets of protein-protein interactions. Nature. 417, 399-403 (2002).
  3. Chin, J. W., Martin, A. B., King, D. S., Wang, L., Schultz, P. G. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. PNAS. 99, 11020-11024 (2002).
  4. Farrell, I. S., Toroney, R., Hazen, J. L., Mehr, R. A., Chin, J. W. Photo-cross-linking interacting proteins with a genetically encoded benzophenone. Nat. Methods. 2 (5), 377-384 (2005).
  5. Wittelsberger, A., Mierke, D. F., Rosenblatt, M. Mapping ligand-receptor interfaces: approaching the resolution limit of benzophenone-based photaffinity scanning. Chem. Biol. Drug Des. 71, 380-383 (2008).
  6. Ieva, R., Tian, P., Peterson, J. H., Bernstein, H. D. Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain. PNAS. 108, 383-391 (2011).
  7. Wang, F., Robbins, S., Guo, J., Shen, W., Schultz, P. G. Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis. PLoS One. 5, (2010).
  8. Deiters, A., et al. Adding amino acids with novel reactivity to the genetic code of Saccharyomycescerevisiae. J. Am. Chem. Soc. 125, 11782-11783 (2003).
  9. Young, T. S., Ahmad, I., Brock, A., Schultz, P. G. Expanding the genetic repertoire of the methylotrophic yeast Pichiapastoris. Biochemistry. 48 (12), 2643-2653 (2009).
  10. Hino, N., et al. Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid. Nat. Methods. 2 (3), 201-206 (2005).
  11. Liu, W., Brock, A., Chen, S., Chen, S., Schultz, P. G. Genetic incorporation of unnatural amino acids into proteins in mammalian cells. Nat. Methods. 4 (3), 239-244 (2007).
  12. Leyton, D. L., Rossiter, A. E., Henderson, I. R. From self sufficiency to dependence: mechanisms and factors important for autotransporter biogenesis. Nat. Rev. Microbiol. 10 (3), 213-225 (2012).
  13. Ieva, R., Bernstein, H. D. Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane. PNAS. 106, 19120-19125 (2009).
  14. Pavlova, O., Peterson, J. H., Ieva, R., Bernstein, H. D. Mechanistic link between barrel assembly and the initiation of autotransporter secretion. PNAS. 110, (2013).
  15. Harlow, D., Lane, D. Using antibodies: a laboratory manual. , Cold Spring Harbor Laborator Press. Cold Spring Harbor, NY. (1999).
  16. Barnard, T. J., Dautin, N., Lukacik, N., Bernstein, P., Buchanan, S. K. Autotransporter structure reveals intra-barrel cleavage followed by conformational changes. Nat. Struct. Mol. Biol. 14 (12), 1214-1220 (2007).
  17. Akiyama, Y., Ito, K. SecY protein, a membrane-embedded secretion factor of E. coli, is cleaved by the ompT protease in vitro. Biochem. Biophys. Res. Commun. 167 (2), 711-715 (1990).
رصد الجمعية ليفرز البكتيرية الفوعة عن طريق عامل يشابك بمواقع محددة
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Cite this Article

Pavlova, O., Ieva, R., Bernstein, H. More

Pavlova, O., Ieva, R., Bernstein, H. D. Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking. J. Vis. Exp. (82), e51217, doi:10.3791/51217 (2013).

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