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

Receiving Cell

Signaling Cell

Ligand

A B C ICN

Ligand-binding Region

Negative regulatory region (NRR)

HD Domain

LNR Domain

A B C HD-N HD-C

S2 S3

Nucleus

Notch Activation I. Ligand binding

II. Regulated cleavages

III. Release of intracellular notch/ Regulation of gene transcription

S1

Structural View of NRR

A B C HD-N HD-C ICN

Negative regulatory region (NRR)

S1 S3S2LNRs are important for maintaining the receptor in

its resting conformation prior to ligand binding.

A B C HD-N HD-C

S1 S2 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 expr

ession system. Vardar, North, Sanchez-Irizarry, Aster, Blacklow (2003) Nuclear Magnetic Resonance Structure of a Prototype Lin12-Notch repeat Modules fro

m Human Notch1. American Chemical Society (42)7061-7067

Research Protocol: Optimizing Recombinant Protein

Expression in Escherichia Coli

CaCl2 competent cells

1 Transformation

Monitor optical density

Inoculate culture with single colony

Grow at 37o C

Collect hourly Samples for

4 hours

Induce with 0.5, 0.1 mM IPTG at 0.5 and0.8 OD

2

34

566x 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 promoterTarget gene

T7 terminator His-Tag

Lac I

Vector: pET15

Induction: IPTGProtocol

Overview:

Competent cells

:::::Transformation :::::Inoculation :::::Induction :::::Purification :::::Gel

Repressorlac 1

T7 RNA polymerase

Lac Operon

E coli

mRNA

T7 Promotor Operator

Target genes

IPTG

IPTG

Protocol Overview:

Competent cells

:::::Transformation :::::Inoculation :::::Induction :::::Purification :::::

Gel

Results Expected outcome:

Molecular

Weight Uninduced 1 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 37o C and induced with 0.1, 0.5mM IPTG at 0.5, 0.8OD.

DiscussionPossible 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. te

mperature, media contents, etc.) difficult for drastic improvement

Inclusion bodies has proven to work previously

mRNA

T7 RNA polymerase

E. coli

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