practical course receptor biochemistry 2019 filepractical course receptor biochemistry 2019...

Practical Course Receptor Biochemistry 2019

Preliminary scheduling for the practical course Receptorbiochemistry. Due to the work

with living cells changes might become necessary if cell growth is not as expected.

Explicit time schedules will be arranged individually.

If you have problems/questions please contact Dr.Karin Mörl [email protected]

Responsible Person: Dr. Karin Mörl

Supervision: Dr. Karin Mörl

Victoria Behr

Kristin Löbner

Sarina Rudolf

Corinna Schüß

Isabelle Ziffert

Einführung für Bock 1 und 2: 3.4.2019, 10:30, Seminarraum 4.Stock, Brüderstr.34

mailto:[email protected]

Experiments planned

Mutagenesis 2 Signaltransduction Assays: Ca2+ Assay and Reporter Gene Assay Receptor Internalization and Recycling (Microscopy, Immunostaining)HiBiT-Tag for surface receptor quantification Receptor Dimerization (BiFC)

Protocols

• One protocol per group

• Protocol has to be signed by each person in the group

• Short introduction, materials & methods only when changed, results, discussion.

• Submission: not later than

Block I: 06.05.Block II: 27.05.

You have to follow basic principles and guidelines for working safely in LaboratoriesIf you work with hazardous materials you have to know the corresponding „Risiko- und Sicherheitssätze“, risk and safetysentences which can be found here: http://gestis.itrust.de/nxt/gateway.dll/gestis_de/000000.xml?f=templates&fn=default.htm&vid=gestisdeu:sdbdeuEven in small amounts, you have to label hazardous materialPoisonous substances have to be shut awayYou have to wear a lab coat. You have to wear gloves and protective glasses when needed. Do not touch labware, equipment , door handles etc. with contaminated glovesHandle hazardous material in a fume hoodKeep working benches clean and tidyKeep hazardous waste as small as possible and discard appropriatelyDon‘t eat and drink in the labsExperimental work is only allowed when supervisor is presentBe informed on directions for use of lab equipment, ask before you use itIf you realize a defect, immediately tell the supervisorDon‘t use gas and air supplying devices in improper wayChemical burns and burns: Rinse with cold water immediately for at least 10 minandIn case of emergency ifor supervisor, dispatcher number on each telephoneFire-extinguisher are to be used all-purposeSmall injuries have to be documented by supervisors for insurance reasonsPregnancy should be communicated as early as possibleOnly students taking part in the course are allowed to enter the labHandle sharps carefully (scalpels and cannula) and discard properly in special containersDon‘t use open fireBe informed on position of fire-extinguisher, emergency showers, electricity emergency stop buttons, alarm signals andescape routes

Instructions for safety working

S1: Work with agents that usually pose a minimal potential threat to laboratory workers and the environment and do not consistently cause disease in healthy adults. Standard microbiology practices are usually enough to protect laboratory workers and other employees in the building.

S2: Work with agents associated with human disease, in other words, pathogenic or infectious organisms posing a moderate hazard. Therefore, because of their potential to cause human disease, great care is used to prevent percutaneous injury (needlesticks, cuts and other breaches of the skin), ingestion and mucous membrane exposures in addition to the standard microbiological practices of S1. Access to the lab is more controlled than for S1 facilities.

Safety level

Safety levels for working with biological material

S1: • well defined working area, door with viewing window, door opening to the outside• Prohibition of food, drink and smoking materials in lab setting• Windows closed while working• Chairs, floor and working benches easy to disinfect (e.g. no wood or cloth)• Daily decontamination of all work surfaces when work is complete• Sink with soap and disinfectant dispenser• Safety cabinets• Mechanical pipetting only (no mouth pipetting allowed)• Safe sharps handling• Avoidance of splashes or aerosols• Label labware (flasks, plates etc.)• If possible you have to use plastic ware with screw caps , especially for storage• Personal protective equipment, such as; eye protection, gloves and a lab coat• Desinfection of waste that got in contact with biological material ( e.g.autoclaving)• immediate decontamination after spills• Only one-way cloth in single-use• disinfect and wash hands after finishing your work• Keep lab clean and tidy, do not store things on cupboards, reduce paper to a minimum• Sinks have to be easily accessible, clean and equipped with eye showers

S2: in addition• Labelling with biohazard symbol• Controlled access• Biological material has to be shut away• Jointless transition from wall to floor• Water tap operatable without hands• Autoclave has to be in the same building• Maintenance repairs only after recorded cleaning, authorization and release• Class II biosafety cabinet• Masks, face shields as needed• Windows always closed

Laboratory Work

Disposal and Transport of Biological Material

• Waste bins or containers have to be labelled „biological material“• in S2 additional biohazard symbol• for genetechnology saying „genetically modified organisms“ • Disposable autoclave bags in containers that are easy to disinfect

• Transport in break proof, disinfectable containers with safe lid that cannot beaccidentally opened

• Transportcontainers have to be labelled „biological material“• in S2 additional biohazard symbol• for genetechnology saying „genetically modified organisms“

Please no cell phone when experiments are performed or discussed!

History of Cell Culture

1910-1913 Alexis Carrel used explants in plasma clots, he subcultured explants by cutting clots and used a mixture of serum, salt solution and chick embryo extract as „plasmatic medium“

Safety class 1 Safety class 2

Protection of product

Kultur tierischer Zellen, S.J. Morgan; D.C. Darling, 1994

Protection of product and scientist

vertical

Sterile Working- Laminar Flow Cabinets

horizontal

Maintains sterile working areaAir blows directly at the investigatorEnough to protect your cultures but not youused for preparation of primary cultures

Air is blown vertically in front of the investigatorA glass barrier is in front of the investigatorAir is recycled and goes through HEPA filter before being returned to the room

Contamination - Mycoplasma

Mycoplasma:

Bacteria

Smallest self replicating organism (0.3-0.8 µm)

Lack cell wall, simple plasma membrane

Occuring extra and intracellularly

pass cell culture filters

Cannot be seen under normal magnification

No overt effects in culture, only subtle ones

The only way to confirm mycoplasma contamination is by routine testing (staining, PCR)

Contamination - Mycoplasma

Mycoplasma can cause:

Changes in Cell growth characteristics

Changes in Cell metabolism

Alteration of cell wall integrity

Disruption of nucleic acid synthesis

Chromosomal aberrations

Changes in cell membrane antigenicity

Decrease of transfection rates and virus susceptibility

Significant changes in gene expression profiles

Influences on almost all functions of the host cell metabolism

• adipose tissue derived hormone

• 244 amino acids

• molecular weight 30 kDa

• anti-diabetic, anti-inflammatory, anti-atherogenic

• Regulation of glucose levels and fatty acid breakdown

• Transgenic mice with increased adiponectin: reduced adipocyte differentiation and increased energy expenditure

• secreted into the bloodstream, accounts for approximately 0.01% of all plasma protein at around 5-10 μg/mL (mg/L)

N-terminus C-terminus

signalpeptide

variableregion

collagen-likedomain

globular domain

1 17 45 110 244

Kadowaki ,T., Yamauchi, T., Kubota, N., Hara, K., Ueki, K., Tobe, K., J Clin Invest, 2006

Adiponectin

Adiponectin Receptors

• consist of seven transmembrane helices

• N-terminus intracellular, C-terminus extracellular

muscle liver

375 amino acids 386 amino acids

Yamauchi, T., Kamon, J., Ito, Y., Tsuchida, A., Yokomizo, T., Kita, S., et al., Nature, 2003Deckert, C.M., Heiker, J.T., Beck-Sickinger, A.G., J Recept Signal Transduct Res, 2006

AdipoR2

EX

IN

GPCR

EX

IN

AdipoR1

EX

IN

AdipoR adiponectin receptorGPCR G-protein coupled receptor

Adiponectin Function

Adiponectin signaling and function in insulin target tissues, J Mol Cell Biol. 2016 Apr;8(2):101-9.

The Neuropeptide Y Family

NPY family: • Neuropeptide Y • Polypeptide YY (PYY)• Pancreatic Polypeptide (PP)

► 36 amino acids► many Y residues► C-terminally amidated► 7 positions constant among all species► PP-fold ??

NPY: • peripheral and central nervous system• neurotransmitter properties• various physiological functions

--CONH2

mediated by Y receptors

NPY, PYY >> PP PYY, NPY >> PP PP >> PYY > NPY NPY, PYY > PP

THE NEUROPEPTIDE Y SYSTEM - RECEPTORS AND LIGANDS

Cabrele, C. and Beck-Sickinger, A.G. J. Pept. Sci. 6: 97-122 (2000)

Larhammar, D. and Salaneck, E. Neuropeptides 38: 141-151 (2004) hYR human neuropeptide Y receptor NPY neuropeptide Y

PP pancreatic polypeptide PYY peptide YY

hY1R hY2R hY4R hY5R

384 aa 381 aa 375 aa 445/ 455 aa

• class A GPCR

• coupled to Gi/ G0

• general effects:

- cAMP ↓

- modulation of K+ and Ca2+

channels

receptors

ligands

•36 aa

• amidated C-terminus

• homology of NPY PYY (70%)

NPY PP (50%)

cAMP cyclic adenosin monophosphate GPCR G protein-coupled receptor

THE NEUROPEPTIDE Y SYSTEM – LOCATION AND FUNCTION

Y1 in hypothalamus

energy homeostasis

anxiolysis

Y4 in small intestine,

colon, pancreas

gastrointestinal regulation

Y2 in brain and

hippocampus

memory retention

regulation of NPY

secretion (i.a.)

Y5 in hypothalamus,

cortex, olfactory

tubercle

energy homeostasis

CNS periphery

Y1 in heart, lung, vascular

smooth muscle cells,

colon, bone marrow

vasoconstriction (i.a.)

Cabrele, C. and Beck-Sickinger, A.G. J. Pept. Sci. 6: 97-122 (2000)

Parker, E., Van Heek, M. and Stamford, A. Eur. J. Pharmacol. 440: 173-187 (2002)

Michel, M. C. et al. Pharmacol. Rev. 50: 143-150 (1998)

Herzog, H. Eur. J. Pharmacol. 480: 21-29 (2003)

THERAPEUTIC POTENTIAL OF THE HUMAN NEUROPEPTIDE Y RECEPTORS

ObesityEpilepsyBreast carcinomasEndocrine tumorsNeuroblastomasIschemic diseases

NPY System - Selective Ligands to Study Structure andFunction

Table 2: Peptide-derived ligands with selective binding affinity for the NPY receptors Y1, Y2 and Y5

Y1

[Arg6,Pro34]-NPY

Agonist

Söll et al. 2001

[Phe7,Pro34]-NPY

[D-Arg25]-NPY

Mullins et al. 2001[D-His26]-NPY

Des-AA11-18[Cys9(Ac),D-His26,Pro34]-NPY

GW1229 with C-terminal methyl esterAntagoni

st

Balasubramaniam et al. 20

01GW1229 with peptide bond between

Arg35-Tyr36 replaced with ψ(CH2-NH)

Y2

[Ala25]-NPY

Agonist

Eckard and

Beck-Sickinger 2001[Ala27]-NPY

NPY13-36 Beck-Sickinger and Jung

1995

[Ahx5-24]-NPY Beck et al. 1989

cyclo-(2/30)[Ahx5-24,Glu2,Lys30]-NPY Beck-Sickinger et al. 1992

Y5

[D-Trp34]-NPY

Agonist

Parker et al. 2000

[Ala31, Pro32]-NPY Cabrele et al. 2002

[Ala31,Aib32]-NPY Cabrele et al. 2000

[cPP1-7,NPY19-23,Ala31,Aib32,Gln34]-PP Cabrele et al. 2000

BREAST CANCER AND Y-RECEPTORS

Breast cancer:

• most common malignancy affecting women

• worldwide: > 1 000 000 cases annually

Stewart, B. W. (2003), WHO World Cancer Report

Breast tumors:

• Y1-receptor expression: > 90 %

• metastases: 100 % Y1-receptors

• normal breast tissue: Y2- receptors

Neoplastic transformation:

• switch from Y2- receptor expression to

Y1- receptor expressionReubi, J. C (2001), Cancer Res. 61(11):4636

Y1R: human Y1 receptor ; Y2R: human Y12receptor

• Coupling of toxic compounds

to hY1 receptor selective NPY

analogs, trojan horse strategy

Breast Cancer Cell

Y1R

N

OAc

N

S

NH

O

OH

O

HN

N

O

O

Cytolysins

TARGETING OF TUMOR CELLS USING NPY CONJUGATES

Specific uptake of toxicsubstances in Cells with high receptor density

TOLL-like Receptors (TLR)

TLRs receive their name from their similarity to the protein coded bythe Toll gene identified in Drosophila in 1985 by Christiane Nüsslein-Volhard. The gene in question, when mutated, makes the Drosophilaflies look unusual, or 'weird'. The researchers were so surprised thatthey spontaneously shouted out "toll" which is the German word for"great" or "amazing".

•Interleukin-1 Receptor/Toll-Like Receptor Superfamily•present in vertebrates, as well as in invertebrates•key role in the innate immune system•most ancient, conserved components of the immune system•single membrane-spanning non-catalytic receptors•recognize structurally conserved molecules that are broadly sharedby pathogens•activate immune cell responses

• Tissue specific / spatiotemporal Expression

Specific Antibodies

Selective Ligands: exchange of single or multiple amino acids (L-Ala Scan, D-Isomer Scan, etc.)

Limiting conformation/flexibility by introducing special amino acidsIntroduction of spacers, cyclization, modification of amino-acidsC- oder N-terminal shortening

• Characterization of ligand-receptor-interaction

Structure-Affinity- und Structure-Activity Studies

• Signal Transduction pathways and subcellular receptor trafficking

• Therapeutic approach

Agonists/Antagonists - Selectivity, stability, oral viability

coupling to toxic substances or markers(e.g. changed receptor subtype expression in breast cancer)

Studies on Receptor Systems

Do cell lines exist, which express the receptor endogenously?

Studies on Ligand-Receptor Interaction: Binding assay

Studies on Signal transduction/ Signalling cascades

Endogenous Signaling pathways – z.B. Phosphorylation Assay, cAMP-Assay, IP3-Assay

Reporter Systems – facilitated Detection and Quantification of Signal Transduction

Changing Signalling Cascades; e.g. Chimeric G proteins

Studies on subcellular Localisation of Receptors:specific Antibodies, specific Ligands

Testing of changed/modified Ligands


Do cell lines exist, which don‘t express the receptor endogenously?

Testing and Analysis of Receptor Mutants:

Transfection of cells with expression plasmids for Receptor/ Receptormutant

Specific labelling of Receptorproteins by Construction of Fusionsproteins (z.B. GFP, HA-Tag, etc.), Check Functionality and Expression!

Do the cells express proteins of the corresponding signal transduction pathways?

Do we need a cotransfection of signalling proteins?

Can we use reporter systems to facilitate analysis and quantification of signalling?


Transfection of Cells

Transfection:•transfer of DNA in eucaryotic cells•DNA mostly in form of plasmids

Transient Transfection: •plasmid is not integrated in genome•gets lost by degradation and cell division•Transfection efficiency (% of transfected cells) depends on method and cell type

Stable Transfection: •DNA is integrated in genome (efficiency in most cases below 0.1%). •linearization of plasmid before transfection enhances integration in genome•isolation of stably transfected clones by antibiotic selection•to maintain the integrated DNA: exposure to antibiotic selection

Tagging Receptors for Visualization andQuantification

Autofluorescent Proteins• genetically encoded fluorescent tags• GFP: 238 amino acid residues (26.9 kDa), exhibits bright green fluorescence

when exposed to light in the blue to ultraviolet range

Small Peptide Tags• Detection with antibodies; e.g. HA-Tag (9 AA)• Tetracysteine-Based Protein Detection e.g. FlAsH-Tag (6 AA);• Enzyme based tags; e.g. SNAP-Tag (182 AA)• Complementation Assays; e.g. HiBiT-Tag (11 AA)

A typical expression vector includes at least 4 key elements:•Bacterial origin of replication (ori)•Antibiotic selection cassette for E. coli (e.g., conferring resistance to ampicillin, kanamycin)•The gene of interest (GOI) expression cassette (including a promoter and a gene-termination or poly(A) signal)•Antibiotic selection cassette for the eucaryotic cell(e.g., conferring resistance toblasticidin S, Geneticin Selective Antibiotic, hygromycin B)

Additional elements may include:•Multiple cloning site (MCS) •Epitope tags•Secretion signal•Protease recognition sites•Internal ribosome entry site (IRES)

Eucaryotic expression vectors

Experiment 1 – Receptor Internalization

REGULATION OF RECEPTOR SIGNALING

nucleus

α β α β

G protein-mediated

signaling

GRK

P P

G protein

agonist

GPCR

AP2 - adaptor protein complex 2; βarr - beta arrestin; EEA1 - early endosome antigen 1; Erk1/2 - extracellular regulated kinase; GRK - GPCR kinase; Ph – phosphatase; Rab - ras-related in brain; Ub – ubiquitin

nucleus

sorting

endosome

Rab5EEA1

Rab5EEA1

α β α β

G protein-mediated

signaling

GRK

P P

βarr

βarr

P P

βarrP PP P

P P

G protein

agonist

GPCR

βarrP P

AP2 clathrin

early endosome

Erk1/2

Rab4

AP2 - adaptor protein complex 2; βarr - beta arrestin; EEA1 - early endosome antigen 1; Erk1/2 - extracellular regulated kinase; GRK - GPCR kinase; Ph – phosphatase; Rab - ras-related in brain; Ub - ubiquitin


nucleus

lysosome

sorting

endosome

Rab5EEA1

Rab5EEA1

Rab7

α β α β

G protein-mediated

signaling

GRK

P P

βarr

βarr

P P

βarrP P

E3

Ub

Ub

Ub

P P

P P

G protein

agonist

GPCR

βarrP P

late endosome

AP2 clathrin

early endosome

Erk1/2

Rab4



nucleus

lysosome

sorting

endosome

recycling endosome

Rab5EEA1

Rab5EEA1

Rab4

Rab7Rab11

Rab11

α β α β

G protein-mediated

signaling

GRK

P P

βarr

βarr

P P

βarrP P

E3

Ub

Ub

Ub

P P

P P

G protein

agonist

GPCR

Ph

βarrP P

late endosome

AP2 clathrin

early endosome

Erk1/2

Rab4



Impact of internalization on signaling

Desensitization of receptors Resensitization of receptors Degradation of receptors Different signalling pathways

Regulation of receptor density in the cellmembrane

Impact on therapeutical applications(e.g. biased ligands, cancer therapy)

TRAFFICKING OF THE HY2 RECEPTOR

New synthesis or reycycling?

Cycloheximide

• fungicide produced by the bacterium Streptomyces griseus

• blocking eukaryotic translational elongation by interfering with the

translocation step in protein synthesis (movement of two tRNA molecules

and mRNA in relation to the ribosome)

Brefeldin A

• inhibits vesicle formation and transport between the endoplasmic

reticulum and the Golgi apparatus

TRAFFICKING OF THE HY2 RECEPTOR

Experiment 2 – HiBiT Tag for surface receptorquantification

HIBIT PROTEIN TAGGING SYSTEM – CLONING VECTOR

IL-6 Signal sequence

HiBiT Tag

YFP

HiBiT-Tag

HiBiT-hY1R-YFPHiBiT-hY2R-YFP

hY1R-YFPhY2R-YFP

To test: Control:

PRINCIPLE OF HIBIT TAG DETECTION

11 AA peptide

Protein

of

interest

(POI)

Contains inactive luciferase subunit (LgBiT)

highly active

luciferase enzyme

Detection limit less than 10-19 moles

COMPARISON HIBIT TO ELISA

Faster

More sensitive

Experiments in live cells

No extensive washing steps

Low background

MEASURE REGULATED PROTEIN EXPRESSION, PROTEIN STABILITY, TARGETED PROTEIN DEGRADATION,

RECEPTOR INTERNALIZATION...

MEASURE LIGAND POTENCY AND EXTENT OF GPCR RECEPTOR

INTERNALIZATION IN A FEW MINUTES

Experiment 3 - Dimerization

Dimerization of GPCR/Adiponectin Receptors

Bouvier et al., Nature Reviews Neuroscience, 2001, 2, 274-286

Bimolecular fluorescence complementation (BiFC)

Dimerization of G-Protein Coupled Receptors

Terrillon and Bouvier, EMBO reports, 2004, 5(1), 30-34

Methods to Detect Dimerization

Western-BlotM

CF-7

SK-N

-MC

HUVEC

hum

aner

Mus

kel

AdipoR1 Monomer

AdipoR1 Dimer

55

MW

- + - + - + - + - +

blocking peptide

74

HEK

293

MCF-7

SK-N

-MC

HUVEC

hum

aner

Mus

kel

AdipoR1 Monomer

AdipoR1 Dimer

55

MW

- + - + - + - + - +

blocking peptide

74

HEK

293

FRET/BRET

Sucrose Gradient

BiFC Bimolecular Fluorescence Complementation

FLAG HA Venus

BiFC Bimolecular Fluorescence Complementation

Transmembrane Helix 5 Adiponectin dependent monomerization

Dimerization of Adiponectin Receptors

Kosel D. et al.; Dimerization of adiponectin receptor 1 is inhibited by adiponectin. J Cell Sci. 2010;123:1320-8.

Experiment 4 – Ca2+ Assay

Efficacy = Describes the way that agonists vary regarding to the response they

produce when they occupy the same number of receptors.

EC50 = The molar concentration of an agonist that produces 50% of the maximum

possible response for that agonist.

Potency = pEC50 = The negative logarithm of the EC50 value.

Emax

2

Emax

EC50

Intrinsic Activity α = Efficacy

Signaltransduction Assay: Dose Response Curves

Log Concentration Agonist

Ko

nze

ntr

atio

n s

eco

nd

mes

sen

ger

signaltransduction assay: dose-

response curves to investigate

the ligand-receptor interactions

EC50

[3H

-In

osi

tolp

ho

sph

ate

]

[Ligand], logEC50

modified ligand increased EC50 value

reduced receptor activity

disturbed interaction

Signaltransduction Assay

Cell Calcium, Volume 59, 2016, Pages 124-134

Ca2+ Signaltransduction

Chimeric G proteins

• chimeric Gqi proteins lead to activation of Phospholipase C increasing

concentration of IP3

activator

inhibition

Gi

[cAMP]

Adenylyl Cyclase

activation

Gq

IP3 + DAG

Gqi

PLC

PiP2

AC

Phospholipase C

Gqi

Ca2+ Assay

X

Ca2+ Assay

Experiment 5 – Reportergene assay

Reportergene Assay: HEK-BlueTM-4 Cells

HEK-Blue™-4 cells are designed for studying thestimulation of TLR4 by monitoring the activation ofNF- κB.

HEK-Blue™-4 cells were obtained by co-transfection of the TLR4, MD2 and CD14 genes and an optimizedSEAP reporter gene (secreted alkaline phosphatase)placed under the control of an NF-κB-inducible promoter into HEK293 cells.

HEK- Blue™-4 cells are highly sensitive to hexa-acylated LPS. They detect as low as 30 pg/ml LPS.

LPS-induced TLR4 activation is readily detected by using QUANTI-Blue™ which turns blue in the presenceof SEAP.

nach Akira (Nature 2004)

TLR4

nach Akira (Nature 2004)

TLR4

HEK-BlueTM-4 Cells

pNiFty plasmids feature an engineered ELAM1 promoter thatcombines five NF-κB sites with the proximal ELAM1 promoter.

This composite ELAM-1 promoter is truly NF-κB specific, on the contrary to the full-length promoter which contains, in additionto three NF-κB sites, an AP1/CREB site that has been shown to influence cytokine induction.

The ELAM1 composite promoter drives the expression of a reporter gene which is induced in the presence of NF-κB and repressed in the absence of the transcription factor.

Two reporter genes are available:- SEAP, the secreted embryonic alkaline phosphatase gene - Luc, the firefly luciferase gene

(InvivoGen 2008)

LPS

TLR

MyD88

p50 p65

IB

NF-Bp50 p65

IB

NF-B

SEAP

5 NF-B-Bindungsstellen5 NF-B-Bindungsstellen

HEK-BlueTM-4 Cells

Cell membrane

Medium

QUANTI-BLUETM Detection of SEAP

(InvivoGen 2008)

QUANTI-Blue™

37 °C 0-2 h

Detektion bei 635 nm

Zellüber-stand

(InvivoGen 2008)

QUANTI-Blue™

37 °C 0-2 h37 °C 0-2 h

Detektion bei 635 nm

Zellüber-stand

LPS-Konzentration:

100 ng/ml

10 ng/ml

1 ng/ml

0 ng/ml

LPS-Konzentration:

100 ng/ml

10 ng/ml

1 ng/ml

0 ng/ml

Dose Response Curve

Expected EC 50 ~0,07 ng/ml

(InvivoGen 2008)

LPS

TLR

MyD88

p50 p65

IB

NF-Bp50 p65

IB

NF-B

SEAP

5 NF-B-Bindungsstellen5 NF-B-Bindungsstellen

Which parameters can be tested?

Cell membrane

Medium

Substance to be tested

Interference with signal transduction

Experiment 6 - Mutagenesis

QuikchangeTM site directed mutagenesis

Finding structurally and functionally important amino acids

Extracellular

Receptor-Ligand Interaction

Intracellular

Interaction with signalling proteins

Point mutationDeletionInsertionChimeric proteins

QuikchangeTM site directed mutagenesis

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