1 / 18

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

zhen
Télécharger la présentation

Biochemical Characterization of LNR_A of Human Notch1 and Notch2

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related