Biochemical Characterization of LNR_A of Human Notch1 and Notch2 Christina Hao

What is Notch? Transmembrane protein receptors of kDa 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)

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

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

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)

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 x His tag Nickel affinity chromatography 7 Run Gel

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

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

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

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?

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.

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

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)

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

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: Sorensen, Mortensen (2004) Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology (115) 2: 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)

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

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