1. Biochemical Characterization of LNR_A of Human Notch1 and Notch2 Christina Hao

2. What is Notch? Transmembrane protein receptors of 300-350kDa Highly conserved Regulates cell growth, differentiation, and cell death in a vast array of tissues through Notch signaling pathway Deregulation of Notch signaling pathway is associated with diseases, eg. Cancer Four mammalian Notch homologs identified (Notch 1-4)

3. Notch Signaling Pathway Receiving Cell Signaling Cell Ligand AB C ICN Ligand-binding Region Negative regulatory region (NRR) HD Domain LNR Domain AB C HD-N HD-C S2S3 Nucleus Notch Activation I. Ligand binding II. Regulated cleavages III. Release of intracellular notch/ Regulation of gene transcription S1

4. Structural View of NRR AB C HD-N HD-C ICN Negative regulatory region (NRR) S1S3S2 LNRs are important for maintaining the receptor in its resting conformation prior to ligand binding. AB C HD-N HD-C S1S2 S3 EGF-like Repeats Gordon, Vardar-Ulu, Histen, Sanchez-Iriarry, Aster, Blacklow (2007) Structural basis for autoinhibition of Notch. Nature: Structural and molecular biology

5. Biochemical Characterization of LNR_A in Human Notch1 and Notch2 Lin12/Notch repeats are structurally independent, disulfide-rich, protein modules of 35 residues.  Can be biochemically characterized in vitro  Requires large amount of proteins for characterization Goal: Optimize protein production in E.coli expression system. Vardar, North, Sanchez-Irizarry, Aster, Blacklow (2003) Nuclear Magnetic Resonance Structure of a Prototype Lin12-Notch repeat Modules from Human Notch1. American Chemical Society (42)7061-7067

6. Research Protocol: Optimizing Recombinant Protein Expression in Escherichia Coli CaCl 2 competent cells 1 Transformation Monitor optical density Inoculate culture with single colony Grow at 37 o C Collect hourly Samples for 4 hours Induce with 0.5, 0.1 mM IPTG at 0.5 and0.8 OD 2 3 4 56 6x His tag Nickel affinity chromatography 7 Run Gel

7. Cell Lines Protocol Overview: Competent cells ::::: Transformation ::::: Inoculation ::::: Induction ::::: Purification ::::: Gel Cell lines Main Features BL21 (DE3) T7 polymerase Lacks two enzymes BL21 (DE3)- PlysS T7 polymerase Lacks two enzymes T7 lysozyme BL21 (DE3)- RIPL T7 polymerase Lacks two enzymes Carry extra genes that recognize mammalian arginine, isoleucine and leucine condons

8. Protocol Overview: Competent cells ::::: Transformation ::::: Inoculation ::::: Induction ::::: Purification ::::: Gel Origin T7 promoter Target gene T7 terminator His-Tag Lac I Vector: pET15

9. Induction: IPTG Protocol Overview: Competent cells ::::: Transformation ::::: Inoculation ::::: Induction ::::: Purification ::::: Gel Repressor lac 1 T7 RNA polymerase Lac Operon E coli mRNA T7 PromotorOperator Target genes IPTG

10. Protocol Overview: Competent cells ::::: Transformation ::::: Inoculation ::::: Induction ::::: Purification ::::: Gel Results Expected outcome: Molecular Weight Uninduced1 hr.2 hr.3 hr.4 hr. 6kda 6kDa Where are the proteins?

11. Conclusion: No significant production of hNotch1 LNR_A was present in E. coli under these experimental parameters: DE3, plysS, RIPL host strains with pET15 vector grown at 37 o C and induced with 0.1, 0.5mM IPTG at 0.5, 0.8OD.

12. Discussion Possible reasons for low expression of target protein: Rapid proteolytic degradation Decreased mRNA stability Toxicity upon induction Unsuitable expression system What is next: Continue experimenting with different conditions (eg. temperature, media contents, etc.)  difficult for drastic improvement Inclusion bodies  has proven to work previously mRNA T7 RNA polymerase E. coli

13. Future Projects  Determine the Ca 2+ affinity of LNRs for other all notch via Isothermal Titration Calorimetry  Test different metals Vardar, North, Sanchez-Irizarry, Aster, Blacklow (2003) Nuclear Magnetic Resonance Structure of a Prototype Lin12-Notch repeat Modules from Human Notch1. American Chemical Society (42)7061-7067

14. Impact of Proposed Projects Information acquired through these studies will: Facilitate the development of structural and functional hypotheses about the regulation of Notch signaling Provide insight into how failure in tight regulation can lead to disease states

15. References Gordon, Vardar-Ulu, Histen, Sanchez-Iriarry, Aster, Blacklow (2007) Structural basis for autoinhibition of Notch. Nature: Structural and molecular biology Sjolund, Manetopoulos, Stockhausen, Axelson (2005). Review: The Notch pathway in cancer: Differentiation gone awry. European Journal of Cancer 41: 2620-2629 Sorensen, Mortensen (2004) Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology (115) 2:113-128 Vardar, North, Sanchez-Irizarry, Aster, Blacklow (2003) Nuclear Magnetic Resonance Structure of a Prototype Lin12-Notch repeat Modules from Human Notch1. American Chemical Society (42)7061-7067 http://www.emdbiosciences.com/product/69661 http://wolfson.huji.ac.il/expression/Bacterial_Strains.htm#strains-exp

16. Acknowledgements Didem Vardar-Ulu Sharline Madera Mentoring in the Science Program Fund Rhulman

17. Questions?

18. Thank You