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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

Deregulation of Notch signaling pathway is associated
with diseases, eg. Cancer

Four mammalian Notch homologs identified (Notch 1-4)
Notch Signaling Pathway
Signaling
Cell
Negative regulatory region (NRR)
Ligand
A B C HD-N
ICN
HD-C
Ligand-binding Region
Notch Activation
LNR Domain
I. Ligand binding
II. Regulated cleavages
HD Domain
III. Release of intracellular notch/
Regulation of gene transcription
S1 S2 S3
Nucleus
Receiving Cell
Structural View of NRR
Negative regulatory region (NRR)
A B C HD-N
HD-C
ICN
EGF-like Repeats
LNRs are important for maintaining the receptor in
S1 S2 S3
its resting conformation prior to ligand binding.
A B C HD-N
HD-C
S1 S2 S3
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)7061-7067
Research Protocol:
Optimizing Recombinant Protein
Expression in Escherichia Coli
CaCl2
competent cells
1
Transformation
2
Inoculate culture
with single colony
Grow at 37o C
3
Run Gel
Monitor optical
density
7
4
6x His tag Nickel
affinity
chromatography
6
Collect hourly
Samples for
4 hours
5
Induce with 0.5,
0.1 mM IPTG at
0.5 and0.8 OD
Cell Lines
Protocol
Overview:
Competent
cells
:::::
Transformation
:::::
Inoculation
:::::
Induction
:::::
Purification
:::::
Gel
Cell lines
BL21 (DE3)
BL21
(DE3)PlysS
BL21
(DE3)RIPL
Main Features
T7 polymerase
 Lacks two enzymes
 T7 polymerase
 Lacks two enzymes
 T7 lysozyme

T7 polymerase
 Lacks two enzymes
 Carry extra genes that
recognize mammalian arginine,
isoleucine and leucine condons

Vector: pET15
Protocol
Overview:
Competent cells
:::::
Transformation
:::::
Inoculation
:::::
Induction
:::::
Purification
:::::
Gel
T7 promoter
Target gene
T7 terminator
His-Tag
Lac I
Origin
Lac Operon
Induction: IPTG
Protocol
Overview:
Competent
cells
:::::
Transformation
:::::
Inoculation
:::::
Induction
:::::
Purification
:::::
Gel
E coli
IPTG
IPTG
mRNA
T7 RNA Repressor
polymerase lac 1
T7 Promotor Operator
Target genes
Results
Protocol
Overview:
Expected outcome:
Molecular
Weight
Competent
cells
:::::
Transformation
:::::
Inoculation
6kda
:::::
6kDa
Induction
:::::
Purification
:::::
Uninduced
1 hr.
2 hr. 3 hr.
4 hr.
Where are the proteins?
Gel
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.
Discussion
Possible reasons for low
expression of target protein:
 Rapid proteolytic degradation
 Decreased mRNA stability
 Toxicity upon induction
 Unsuitable expression system
E. coli
mRNA
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
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
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: 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
Acknowledgements
Didem Vardar-Ulu
Sharline Madera
Mentoring in the Science Program
Fund
Rhulman
Questions?
Thank You