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Biochemical Characterization of LNR_A of Human Notch1 and Notch2

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

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Biochemical Characterization of LNR_A of Human Notch1 and Notch2

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  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. A B C HD-N HD-C S2 S3 Nucleus Notch Signaling Pathway Signaling Cell Negative regulatory region (NRR) Receiving Cell Ligand A B C ICN Ligand-binding Region Notch Activation I. Ligand binding II. Regulated cleavages III. Release of intracellular notch/ Regulation of gene transcription S1 LNR Domain HD Domain

  4. A B C ICN HD-N HD-C A B C HD-N HD-C S3 S1 S2 Structural View of NRR Negative regulatory region (NRR) EGF-like Repeats LNRs are important for maintaining the receptor in its resting conformation prior to ligand binding. S1 S2 S3 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 CaCl2 competent cells Transformation Inoculate culture with single colony Grow at 37o C 1 2 3 Run Gel Monitor optical density 7 4 6x His tag Nickel affinity chromatography Collect hourly Samples for 4 hours Induce with 0.5, 0.1 mM IPTG at 0.5 and0.8 OD 6 5

  7. Cell Lines Protocol Overview: Competent cells ::::: Transformation ::::: Inoculation ::::: Induction ::::: Purification ::::: Gel

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

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

  10. Expected outcome: Molecular Weight Uninduced 1 hr. 2 hr. 3 hr. 4 hr. 6kda 6kDa Results Protocol Overview: Competent cells ::::: Transformation ::::: Inoculation ::::: Induction ::::: Purification ::::: Gel 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 37o C and induced with 0.1, 0.5mM IPTG at 0.5, 0.8OD.

  12. 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 Discussion E. coli T7 RNA polymerase

  13. Future Projects • Determine the Ca2+ 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 Notchsignaling • 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

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