Biochemical Characterization of LNR_A of Human Notch1 and Notch2

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Biochemical Characterization of LNR_A of Human
Notch1 and Notch2

• Christina Hao

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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)

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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
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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
S3
S2
Gordon, Vardar-Ulu, Histen, Sanchez-Iriarry,
Aster, Blacklow (2007) Structural basis for
autoinhibition of Notch. Nature Structural and
molecular biology
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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

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Research Protocol Optimizing Recombinant
Protein Expression in Escherichia Coli
CaCl2 competent cells
Transformation
Inoculate culture with single colony Grow at
37o C
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2
3
Run Gel
Monitor optical density
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4
Collect hourly Samples for 4 hours
Induce with 0.5, 0.1 mM IPTG at 0.5 and0.8 OD
6x His tag Nickel affinity chromatography
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Cell Lines
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
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Vector pET15
T7 promoter Target gene T7 terminator His-Tag
Protocol Overview Competent cells
Transformation Inoculation
Induction Purification
Gel
Lac I
Origin
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Induction IPTG
Lac Operon
IPTG

• Protocol Overview
• Competent cells
• Transformation
• Inoculation
• Induction
• Purification
• Gel

E coli
Repressor lac 1
T7 RNA polymerase
Target genes
T7 Promotor
Operator
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Results
Protocol Overview Competent cells
Transformation Inoculation
Induction Purification
Gel
Where are the proteins?
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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.

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Discussion
E. coli

• Possible reasons for low
• expression of target protein
• Rapid proteolytic degradation
• Decreased mRNA stability
• Toxicity upon induction
• Unsuitable expression system

T7 RNA polymerase

• What is next
• Continue experimenting with different conditions
  (eg. temperature, media contents, etc.) ?
  difficult for drastic improvement
• Inclusion bodies ? has proven to work previously

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Future Projects

• Determine the Ca2 affinity of LNRs for other all
  notch via Isothermal Titration Calorimetry
• Test different metals

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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

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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)
  2113-128
• 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_Str
  ains.htmstrains-exp

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Acknowledgements

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

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Questions?
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Thank You