1. Taylor Bendt Faculty advisor: Dr. Gary Merrill

2. DNA Damage p53 DNA repairApoptosisp21 Cell cycle arrest Genome maintenance  Important for cancer prevention http://www.topnews.in/files/CancerCells22.jpg

3.  If p53 is oxidized Ac92 could have an inhibitory function.  Prevent apoptosis  Prevent cell cycle arrest Insights for preventing cancer DNA Damage p53 DNA repairApoptosisp21 Cell cycle arrest Genome maintenance

4.  A baculovirus is a viral parasite that infects insects  Ac92 is a baculovirus protein that binds FAD and has sulfhydryl oxidase activity  Human p53 expressed from a baculovirus vector copurifies with Ac92 (Ac92 and human p53 can physically interact) http://www.biochem.wisc.edu/faculty/friesen/

5.  Involved in viral assembly, as has been shown for sulfhydryl oxidases of mammalian viruses  defeats the antiviral activity of host cell p53 (mechanisms to defeat p53 are common in mammalian viruses)

6.  Ac92 binds to Sfp53 (the p53 protein of Spodoptera frugiperda – the natural host of baculoviruses)  Ac92 oxidizes Sfp53  Sfp53 is inhibited by oxidation

7.  Ac92 binds Sfp53: co-immunoprecipitation assays  Ac92 oxidizes Sfp53: sulfhydrylhistochemistry  Sfp53 is inhibited by oxidation: target gene activation in transfected insect cells

8.  Screen for recombinant bacteria that overexpress Sfp53 and Ac92 that contain reciprocal epitope tags  Prepare purified epitope-tagged Sfp53 and Ac92  Determine if HA-tagged Sfp53 coprecipitates Ac92 and if Flag-tagged Ac92 coprecipitates Sfp53

9.  Dr. George Rohrmann provided BL21 (E. coli) glycerol stocks  3 clones with HIS tagged HA tagged Sfp53  5 clones with HIS tagged Flag tagged Ac92  Clones also contained kanamycin resistance  Cells are grown in liquid medium containing kanamycin to kill all non-transformed bacteria

10.  The proteins Sfp53 and Ac92 should only be produced in the presence of IPTG  Comparing induced and uninduced protein products should confirm that the clones work

11. i 66 45 31 21.5 14 6.5 Sfp53 Ac92 Sfp53 Old Ac92 ii

12. M.W Markers pET28 – Ac92 (old) pET28a-sfp53 clone 1 pET28a-sfp53 clone 2 pET28a-sfp53 clone 3 pET28 – Ac92 clone 9 pET28 – Ac92 clone 10 pET28 – Ac92 clone 11 pET28 – Ac92 clone 12 + 66 45 31 21.5 14.4 6.5 Sfp53 Ac92 +++++++ ------ IPTG Conclusions: 1) Sfp53 clones are correct (they produce a 46-kD that is inducible by IPTG) 2) Ac92 clones are incorrect (they don’t produce detectable protein) Subsequent sequencing showed the clones had a frameshift mutation.

13.  Grow a 500 mL culture  Break by sonication in the presence of protease inhibitors  Clarify by centrifugation  Bind His-tagged proteins to TALON resin  Wash resin  Elute bound protein with imidazole  Monitor purification by SDS-PAGE, and Coomassie staining http://www.odec.ca/projects/2007/meng7l2/new_page_8.htm

14. 12345678910 52 42 34 26 17 10 72 95 135

15. Uninduced 50 75 100 150 250 37 25 20 15 10 Sfp53 Induced H.S. Pellet H.S Sup. Unbound 10 mM Imid. Eluate 1Eluate 2Eluate 3Eluate 4Eluate 5 M.W Markers

16. Uninduced Induced H.S. Pellet H.S. Sup. Unbound 10 mM Imid. Eluate 1Eluate 2Eluate 3Eluate 4Eluate 5 Conclusions: Most of the Sfp53 is contained in the high speed pellet, and relatively little is contained in the purified portions. By Lowry, the protein concentration of the most concentrated eluate is less than 1μg/μL, not enough for use. Sfp53

17.  Not enough Sfp53 was purified.  May be trapped in inclusion bodies. http://aem.asm.org/cgi/content/full/69/2/1295

18.  Grow bacteria in 18 ° after induction  Change induction time from 6 hours to 20  Known to work for Ac92 from previous work

19. MW MarkersUninducedInducedH.S. PelletH.S. Sup.Unbound10mM Imid.50mM Imid.100mM Imid.Eluate 1Eluate 2Eluate 3Eluate 4Eluate 5 50 75 100 150 250 37 25 Sfp53 Conclusion: The 18 ° growth did not help solubilize the protein, and no Sfp53 was purified.

20.  A collaborator has managed to produce soluble Sfp53 in BL21 cells  There is one difference in protocol: volume of resuspension of the bacterial pellet before breaking open cells by sonication

21. MW MarkersUninducedInducedH.S. Sup.H.S. PelletUninducedInducedResuspensionH.S. Sup.H.S. Pellet 50 75 100 150 250 37 25 Sfp53 20 15 Conclusion: Even with larger resuspension volume Sfp53 is not solubilized.

22. IPTG - ++++ - - - Sfp53 Clone 3 Ac92 Clone 1 Ac92 Clone 2 Ac92 Clone 3 Ac92 Conclusion : The new Ac92 is correct and produces a detectible amount of protein when induced.

23.  The correctness of these clones is also supported by an orange color generated when the protein binds to TALON resin

24. MW MarkersUninducedInducedH.S. PelletClarified LysateUnbound10 mM Imid.Eluate 1Eluate 2Eluate 3Eluate 4Eluate 5 50 75 100 150 250 37 25 15 20 10 Ac92 Conclusion: Ac92 was successfully purified using TALON RESIN.

25.  Receive Sfp53 from collaborator  Using co-immunoprecipitation detect interaction  Test for oxidizing activity of Ac92 on Sfp53

26.  Dr. Gary Merrill  Dr. George Rohrmann  HHMI  Frances Cripps  URISC  Kevin Ahern http://www.biochem.wisc.edu/faculty/friesen/