Protein Overexpression. Overnight cultures (15 mL) of transformed Escherichia coli BL21 expressing GST (glutathione S-transferase)-peptide, GST-EBNA1(Epstein-Barr nuclear antigen-1) peptide and 6xHis-USP7 peptide required 45 mL of LB Amp+ Km+ medium dispensed into GST and 6xHis-USP7 cultures while LB Amp+ medium was dispensed into the GST EBNA1 culture. Three flasks each with a bacterial culture was placed on a shaking incubator (250 rpm) for 1 hr at 37°C for optimal cell growth. An optical density reading was obtained from a 1 ml sample from each culture after incubation using a spectrophotometer set at 600 nm to assure the appropriate cell density was achieved before induction. Peptide expression was induced by exposing the culture to 0.55 mL of 0.1 M IPTG and then incubated again for 1 hr at 37°C in the shaking incubators. 0.15 mL of the non-induced GST-EBNA1 bacterial culture was centrifuged (13000 rpm) for 2 min at room temperature (RT), in which the pellet was stored at -20°C to use as sample 1 for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. The remainder of the induced culture was centrifuged using Sorvall Legend RT (4000 rpm) for 20 min at 4°C. The wet pellets were put away after centrifugation at -20°C.
Protein Extraction and Purification. The USP7 bacterial pellet was thawed at RT, 1 mL of cold Ni 2+ Lysis/Binding Buffer (5 mL of Common Buffer + 5 uL of 5 M Imidazole, which had an Imidazole concentration of 5 mM) was added to the bacterial pellet and thoroughly vortexed. The cells were lysed using the sonicator (30% intensity, 4 x 20 sec pulses, and 15 sec gaps).
Unbroken cells and insoluble proteins were sediment using the refrigerated micro-centrifuge (...
... middle of paper ...
...ed and further studies will be necessary to determine what the interaction affects or how the two proteins interact.
Department of Biology 2013, BIOL 2070 Research Methods in Cell and Molecular Biology, Resource Manual, pp 98-124.
Frappier, L. 2013. EBNA1 and Epstein-Barr virus associated tumours. New York, NY: Springer.
Holowaty, M. N., Sheng, Y., Nguyen, T., Arrowsmith, C. and Frappier, L. 2003. Protein interaction domains of the ubiquitin-specific protease, USP7/HAUSP. Journal of Biological Chemistry, 278 (48), pp. 47753--47761.
Holowaty, M. N., Zeghouf, M., Wu, H., Tellam, J., Athanasopoulos, V., Greenblatt, J. and Frappier, L. 2003. Protein profiling with Epstein-Barr nuclear antigen-1 reveals an interaction with the herpesvirus-associated ubiquitin-specific protease HAUSP/USP7. Journal of Biological Chemistry, 278 (32), pp. 29987--29994.
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