Approaches

¾ Yeast two-hybrid system

¾ Tandem affinity purification (TAP) and
mass spectrometry (MS)
Protein-protein interaction ¾ Immunoassay
¾ Protein chip
¾ Biosensor system
¾ Molecular image

Screening for interacting protein (bait and prey)

TCL
AKT kinase

apoptosis
P
P

TCL: protooncogene

T-cell leukemia

Strong promotor
(T cell receptor promotor)

TCL
TCL
AKT kinase
Unknown mechanism

apoptosis
P
T-cell leukemia P

1
 Akt: a kinase promoting cell survival Screening for interacting protein (bait and prey)
Growth factor TCL
AKT kinase

Apoptosis

Yeast two-hybrid system

Method 1:

Yeast two-hybrid

Model of activation of Gene Transcription Model of activation of Gene Transcription

上游基因

Activatior proteins
Transcription factors
RNA polymerase

Activatior proteins
AD
DB
下游基因
下游基因

2
 Activatior proteins
Activatior proteins
AD 上游基因
DB
下游基因

Gal4 protein

DB: DNA binding domain

AD: Activation domain 下游基因
LexA protein

Chimeric activation protein Chimeric activation protein

Gal4 protein
Gal4 protein
LexA protein
LexA protein

上游基因
Fusion protein
(Chimeric protein)
Restriction
enzyme

上游基因 上游基因

ligase

Chimeric m-RNA

Chimeric activation
protein

3
Chimeric activation protein 上游基因 上游基因

Gal4 protein

下游基因
Gal4 AD
LexA BD

Gal4 AD
LexA BD

上游基因 activation binding

DB AD

DB: DNA binding domain

AD: Activation domain
AD

DB

下游基因

上游基因

外來基因-1 外來基因-2

prey bait

下游基因

報告基因

4
 Screening for interacting protein (bait and prey)

bait
DNA extraction
DNA sequencing

上游基因 上游基因

外來基因-1 外來基因-2 外來基因-1 外來基因-2

prey bait prey bait

c-DNA library
人類
m-RNA
細胞
報告基因未表現

Result 1:
prey
bait
Yeast two-hybrid
報告基因表現

5
 AKT kinase Method 2:
TCL

Yeast two-hybrid
Domain scan

Identification of interacting domain (bait and prey)

TCL
AKT kinase TCL

AKT kinase

上游基因
AKT TCL片段

外來基因-1 外來基因-2

prey bait
AKT kinase

DNA fragments
TCL
DNA TCL
表現強度
片段

6
 Result 2:
Yeast two-hybrid
Domain scan

Confirmation of interacting protein (bait and prey)

Method 3:
Coimmunoprecipitation

Coimmunoprecipitation Gel electrophoresis Western blotting

B B

7
Western blotting

- -
1. safety lid
2. cathod assembly with latches
3. filter paper
4. gel
5. membrane
6. filter paper
7. anode platform
+
+ 8. power cable
9. base

Tagged recombinant protein

Gel electrophoresis Western blotting
基因

transfection

Tagged recombinant protein Tagged recombinant protein

Anti-tag antibody

transfection

8
 Confirmation of interacting protein (bait and prey)

Gel electrophoresis Western blotting

Double transfection of two tagged genes

tag1 基因1

tag2 基因2

transfection transfection

Western blotting Western blotting

Gel electrophoresis Gel electrophoresis

Anti-tag1 antibody Anti-tag2 antibody

9
 Result 3:
Coimmunoprecipitation

AKT-HA tag
TCL1-myc tag

AKT-HA tag TCL1-myc tag

Regulation of apoptosis
Fas
2
FADD 2
3 Caspase 8 FADD
IAP: inhibitor of apoptosis 3 Caspase 8
Caspase 9
Caspase 9
Caspase 3 baculovirus
Apaf-1 Caspase 3
Apaf-1
Caspase 6 drosophila
1 DIAP
Cyt.C 1 Caspase 6
proapoptic signal Cyt.C
(Grim,Reaper,HID)
1. Bcl-2
2. FLIP mammalian
3. IAP MIHA
(mammalian IAP homolog A)

10
drosophila
DIAP Fas
proapoptic signal
2
FADD
(Grim,Reaper,HID)
3 Caspase 8
mammalian Caspase 9
MIHA Caspase 3
?????????? Apaf-1

1 Caspase 6
Cyt.C

Flag-MIHA (IAP)

HA-DIABLO

11
 1. Preactivated Surface 2. Bind ‘Capture’ Molecule 3. Block unused sites

Method 4:
4. Analyte Capture 5. Wash 6. Add Matrix

SELDI-TOF

Laser
Pulses

Protein-protein interaction

AKT-HA tag

Result 4:
SELDI-TOF

12
 Practical Considerations for
Protein-Protein

TCL
AKT kinase

apoptosis
P
P

Molecular Recognition Studies

‰Antibody-antigen binding
‰Directly coupled or captured on Protein A/G
1. Preactivated Surface 2. Bind ‘Capture’ Molecule 3. Block unused sites
‰Receptor-ligand interaction
‰Protein-protein

‰Protein-small molecule
‰‘Fishing’ for binding partners – domains or intact ‘bait’ 4. Analyte Capture 5. Wash 6. Add EAM

‰DNA/Protein or RNA/Protein interaction

Laser
Pulses

Ligand Enzyme
Antibody Protein A/G Receptor DNA

‰Carbonyl Diimidazole (CDI) activated amine surface

Basic Experimental Procedure PS10 ‰Covalently Binds Proteins/Peptides through amine group
‰Pre-wet spots ‰Immobilization conditions
‰pH 8-9
‰Covalently attach the capture molecule
O ‰med - high salt
‰ 1-2 hr at RT, or overnight at 4° C N ‰Washing conditions
N
H

‰Block unreacted sites (ethanolamine, Tris, etc.) O N ‰Buffer with detergent

(eg PBS 7.2 with TX100)
Wash to remove unbound capture molecules
N N
‰ H
N

‰Capture of specific protein from solution protein

‰ 15 min - 2 hr at RT, or overnight at 4° C
O

‰Wash to remove N N
H
Add EAM
H
‰ O

‰Analyze N
H
N
H

13
 Epoxide activated amine surface
PS20 ‡

‡ Covalently Binds Proteins/Peptides through amine group

‡ Immobilization conditions
‡ pH 8-9
‡ med - high salt
O
‡ Washing conditions
‡ Buffer with detergent
O
(eg PBS 7.2 with TX100)

protein

OH
NH

OH
NH

Antibody Protein A/G

pre-
pre-coupled with Protein G pre-
pre-coupled with Protein G

Antibody Protein A/G

‰Can use antibodies in the presence of other proteins, Tris, azide, etc.
‰Antibodies on the surface all in same orientation [signal may be higher]
‰Antibody will “fly” if not covalently crosslinked
[Antibody peaks at 150, 75, 48, and 22 KDa]

Experimental Parameters
PS10 vs. PS20 ‰ Keq
PS10 Arrays
‰ kd
‰Higher capacity ‰ Solution conditions [pH, salt concentration, etc.]
‰Higher non-specific binding
‰Use higher detergent (typically 0.5% Triton X-100)
‰Longer washes
‰Hydrophobic background

PS20 Arrays
1. Preactivated Surface 2. Bind ‘Capture’ Molecule 3. Block unused sites

‰Lower capacity
‰Lower non-specific binding
‰Use lower detergent (typically 0.1% Triton X-100)
‰Shorter washes 4. Analyte Capture 5. Wash 6. Add EAM
‰Hydrophilic background

14
Experimental Parameters – Sample Binding Experimental Parameters – Washing
A + B AB a non-
non-equilibrium process
[AB]
Keq = = ka/kd
[A][B]
+
‰Keq, ka, and kd all depend on solution conditions
‰Reproducible binding requires reaching equilibrium in the
Wash
binding step

+
+

Experimental Parameters – Washing Good vs. Bad Antibodies

a non-
non-equilibrium process

+ Intact Ab +1
light chain50000 100000 150000
40
Intact Ab +2 147749.8+H
30 23555.3+H

20 heavy chain 74204.5+H

Albumin dimer
10
49607.4+H 120000 130000 140000 150000 16000
0
‰Washes should be done for as short a time as possible.
133419.3+H
66994.7+H 10
75
Albumin +1 7.5 Intact Ab
‰Washing at 4oC will decrease loss of specific binding. 50
Albumin +2 5
148021.7+H
2.5
25 33600.2+H
133419.3+H 0
0 148021.7+H
120000 130000 140000 150000 16000
0.4 147568.0+2H
0.3 147568.0+H
0.2
0.1
0

50000 100000 150000

15
16
 Epitope mapping

Antibody directed enzyme prodrug therapy

(ADEPT)

antibody directed enzyme prodrug therapy Protein and peptide therapeutics：

(ADEPT) Induce an antibody response
cytotoxic drug
A prodrug 1. foreign proteins
2. recombinant human proteins

Knowledge of the amino acid sequence of a protein allows

antitumor antibody CPG2 identification of linear epitopes

1. reaction of overlapping peptides with antibody.

2. Mutations based on this strategy have been used
successfully to give clinically valuable reduction in
immunogenicity
Carcinoembryonic antigen (CEA)
colorectal tumors

17
 CEA is a tumor selective marker
Epitope Gastrointestinal carcinomas
Breast
lung
ovarian carcinomas

1. linear peptides
2. Conformational epitopes Anti-CEA Ab

Anti-enzyme Ab

Carcinoembryonic antigen (CEA)

colorectal tumors
repeated therapy was prevented by immunogenicity

ADEPT: chemical conjugation new generation of ADEPT:genetic engineering

greatly reduced immunogenicity
antitumor antibody CPG2 genetically manipulated to reduce immunogenicity

chemical conjugation Cannot be recognized by Ab

new generation of ADEPT:genetic engineering

greatly reduced immunogenicity
genetically manipulated to reduce immunogenicity mutation

CPG2
MFE-23 (anti-CEA
sFv） Anti-CEA Ab
MFE-23::CPG2 fusion protein
Anti-enayme Ab

Prodrug Phase I ADEPT clinical trial

mutation
CEA CM79
Phage Ab

18
 A
Full-length APP E

Trypsin digestion
B

Peptides mixture M/z

MALDI-TOF MS
Inteact with
ProteinChip
Peptide profile for APP

C D Captured peptide F

APP-interacting
protein
M/z
SELDI-TOF MS
ProteinChip for mapping the epitope

G
Peptide sequencing

Limited Proteolysis as a Probe of

Higher-Order Structure

Mapping a Protein/DNA interface 3D crystal structure of the Max/DNA

1. observed fragment masses, complex
2. known primary sequence,
3. known protease sequence-specificity

19
 Surface Plasmon Resonance
--- SPR is a real-time, label-free, optical detection method
for studying the interaction of soluble analyte with
Surface Plasmon Resonance immobilized ligand
Biochips --- completely non-specific

(表面電漿共振 生物晶片) 1902 Wood – anomalous diffraction due to excitation of

surface plasma waves
1968 Otto – the method of attenuated total reflection
1988 (1990) BIAcore

Total Internal Reflectance Total Internal Reflectance

n1 > n2 n1 > n2 n1 > n2

n1 < n2

Total Internal Reflectance Thin Conducting Film

n1 > n2 evanescent distance ± 100 nm A medium of high refractive index
A thin layer with good electric conductivity
A medium of low refractive index

20
 SPR Sensor Classification
Characteristics of SPW
Classification by Coupling Method Optical Fiber SPR Sensor

Gold is the most practical.

(In) too expansive, (Na) violently reactive,
(Cu, Al) too broad in their SPR response
Optical Probe Sensor (White Source)
(Ag) too susceptible to oxidation
I. Stemmler et al. :Sensors and Actuators B 54 (1999) 98–105

Side Polished Fiber Sensor

(Monochromatic Light)
J. Homola et al. :Sensors and Actuators B 54 (1999) 3–15 R. Slavik et al. :Sensors and Actuators B 54 (1999) 74–79

Elements of SPR Sensor System Commercial SPR Sensor System

Optical Device
-Sensitivity/Cost/Patent Biacore® 3000,2000,1000
-Dynamic Range

Biacore® X, J, Q, S51, C
Surface Chemistry Sensor Chip Biochemical
(Immobilization) -Thin Metal Film Contents
Spreeta

Micro-Fluidics
-Continuous Flow of Small Volume
-Automated Liquid Handling
IBIS
Technologies IBIS-
IBIS-I, II
Sensor Signal Processing – Electronics, Software Eng.

BIAcore
BIO-SUPLAR 2

21
 Real-Time Kinetics Sensorgram

SPR Equipment

polarizer Convex lens Photo sensor

laser Sample cell
prism

First rotation stage

Photo detector Second rotation stage

Metal Ion Detection Metal Ion Detection Result

z Thiol group SAM of HDT (1,6-hexanedithiol) Pt ion detection by HDT layer
z Attraction to Au, Pt, Pd, Ag
180

Metal ions 160 Before 1

After 80 min
1,6-hexanedithiol Au layer 140
0. 8
Au 120
Intensity [㎼]

HS
Δ θ /θ

-s 100 0. 6
SH SH
80
-s SH 0. 4
Ag 60
-s SH 40
0. 2
SAM on Au layer -s SH 20

-s 0
SH Pt 0
0 50 100 150 200
55 60 65 70 75 80
-s SH
Angle [degree] Ti me [min]

-s SH Pd
-s SH

22
 DNA Array (1)
DNA Array (2)

* Robert M. Corn, Anal. Chem.2001, 73, 5525-5531 * Robert M. Corn, Langmuir 2001, 17, 2502-2507

Immuno-Sensor
Tao Theory (2) (i) antiprogesterone
(ii) anti-testosterone
(iii) anti-mouse Fc

pH<6 10<pH<6 pH>10

Protonation/ Non-specific binding to target proteins
Deprotonation
0.002
pH10.5 sol.
pH10.5 sol.
0.001

0.000
Δθ

Au substrate
-0.001
oxygen pH3 sol.
sulfur carbon hydrogen bare Au
pH3 sol. Au + SAM
-0.002
− +
RCOOH ←⎯
⎯→ RCOO + H
pKa 0 100 200 300 400 500 600

time (sec)

* S.Chah, Langmuir, 18(2), 314-318

Lab on chip

• Developed a sensor to replace

existing diagnostic tests Identification of
• Miniature
• No need for centralised testing protein-protein interaction
facility, can use for near patient
test
• One step
• Rapid
• Simple

23
 Approaches Principle of the yeast two-hybrid
system
¾ Yeast two-hybrid system 此系統是由 Fields 與 Song 等人在 1989 年建立
¾ Tandem affinity purification (TAP) and
mass spectrometry (MS)
¾ Immunoassay
¾ Protein chip
¾ Biosensor system
¾ Molecular image

Traffic 2000, 1, p764

The distribution of experimental methods for Typical two-hybrid screens

detecting the protein–protein interactions
documented in Database of Interacting Proteins
(DIP: http//dip.doe-mbi.ucla.edu)

Nucleic Acids Research 2001, 29, p239-240 Curr Opin Chem Biol. 2001, 6, p58

Array screens
Matings and two-hybrid tests can be automated when large sets Applications for two-hybrid array
of preys have to be assayed, as in the case of whole genomes.

Curr Opin Chem Biol. 2001, 6, p58 Curr Opin Chem Biol. 2001, 6, p59

24
 Bacteria two-hybrid
The screens of two-hybrid array
¾依分子機轉的運用主要可分為兩大
類 ，其一是基於調節報告基因
(reporter gene) 的轉錄活化或抑制
為主的應用，例如 λ repressor
dimmers、Lex A homodimers、 Ara C
activator 等轉錄調控因子的應用，
其原理類似 yeast two-hybrid系統中
Gal 4 轉錄因子的運用。

Curr Opin Chem Biol. 2001, 6, p59

¾基於活化態酵素的恢復 (reconstitute) 原 Bacteria vs. Yeast system

理之應用。例如 Bordetella pertussis 中，活
化態 adenylate cyclase 需由 T18 與 T25 這兩 ¾優點
段互補片段來構成，因此將有交互作用的 生長快速、容易操作、轉型效率高、避免真核系
polypeptide 分別融合在T18 與 T25 後，轉型至 統中內生性轉錄因子造成偽陽性等。
細菌體內，經形成具有活性的adenylate cyclase
後，將促使 cAMP 的生成，而其訊息傳遞得以 ¾缺點
進行，接著利用細菌生長代謝時，是否可利用 1. 無法展現轉譯後修飾 (post-translational
maltose 或 lactose 作為碳素的來源 (cAMP modification)，折疊正確構型 (conformation) ，
mediated) 的特性，以選擇性培養基，間接得知 高層級結構。
adenylate cyclase 的活化型態，亦即獲知兩兩
蛋白質或polypeptide 之間的交互作用情形 。 2. 研究資料庫不如酵母菌豐富。

Identification of Protein
Complexes

plasmid transfect

Identification of protein
expressing into cells
epitope-tagged
bait protein

complexes by the combination of

tandem affinity purification (TAP) isolate
protein
and mass spectrometry (MS) complex

bait

analyze
components inspect
by mass spec on 1-D gel
(with or without gels)

25
 Protein complexes take the bait
Gene targeting procedure of the yeast system
Many cellular functions are
carried out by proteins that are
bound together in complexes. In
two new large-scale studies,
labelled proteins are used as‘bait’
to capture and identify those
complexes.

Nature 2002, Vol. 415, p142 Nature 2002, 415, p 123-124

Overview of the TAP purification Basic of protein identification

strategy

GENE PRODUCT I.D.

dB
MALDI PROTEIN
PROTEOME MS GENOME
EST

MALDI
MS/MS
DIGEST

ESI
HPLC MS/MS

Methods 2001, 24, p220

Non-Gel Proteomic Example:

Workflow Proposed model of the polyadenylation
•Multiple Dimension Liquid Chromatography (MDLC) machinery

Peptide
Separation
Each fraction
can be
Samples preparation CEX injected onto

MDLC Workstation
LCMS/MS
MALDI/MS/MS
Software

MS/MS System

Nature, 2002, Vol. 415, p143

26
Functional organization of the yeast Systematic Identification of Protein Complexes in
proteome by systematic analysis of protein Saccharomyces cerevisiae by Mass Spectrometry
complexes
Kss1 = MAP kinase Cdc28 complex

Blue arrows = known interactions

Nature, 2002, Vol. 415, p144 Red arrows = new interactions
Nature, 2002,415, p180-
p180-183

Conventional immunoassay Protein chips

and binding assay Three different types of protein microarrays

¾ immunoprecipitation (IP)

¾ co-immuno precipitation (coIP)

¾ pull down assay

¾ in vitro binding assay

Curr Opin Chem Biol. 2001, 5, p40–45

Current immunoassay strategies

used in protein-detecting
microarrays

Multiple fluorophores

nature genetics 2002, suppl., 32, p528

27
 Complications arising from
regulated protein–protein
interactions 應用實例
¾例如微陣列免疫試驗組 (sandwich
microarray-base assay)，其中包括商品
化的細胞激素 (cytokine) 免疫試劑組，
以及100 種以上用來分析細胞內容物
(cell lysates) 的抗體應用，其主要是在探
討特定功能的蛋白質體 (specific
functional proteome)，或是疾病相關的
蛋白質圖譜 (protein profile)。

nature genetics 2002, suppl., 32, p529

Non-immunoassay strategies
Example: Single step generation of protein Example:
arrays from DNA by cell-free expression and A ‘proteome chip’ composed of
6,566 protein samples representing
in situ immobilisation. 5,800 unique proteins, which are
spotted in duplicate on a single
nickel coated glass microscope
slide39. The immobilized GST
fusion proteins were detected using
a labeled antibody against GST.

Nucleic Acids Research 2001, 29, e73

Science 2001, 293, p2101

蛋白質晶片應用上的考量
¾ 固定化法與蛋白質構型的維持

¾大量表現出在各種生理狀態下的蛋
白質 （例如轉繹後修飾蛋白質、活
化態、不活化態與突變態等），並
經自動化純化重組蛋白質，當作晶
片結合的蛋白質基質，是該方法極
需要開發的重點 之一。

28
 表層薄膜共振
Biacore‘s surface plasmon resonance (SPR)
technology What can Biacore do?
SPR allows sensitive detection of molecular interactions
in real time, without the use of labels. ¾ How specific is the interaction?
¾ How fast is the interaction?
How specific is the interaction?

¾ How strong is the interaction?

¾ How much interactant is present?
¾Connect to Mass Spectrometry!

http://www.biacore.com/technology/spr_technology.lass

Fluorescence resonance energy transfer (FRET)

分子影像學 Molecular image can be used to analyze protein–protein interaction
dynamics in living cells.
¾ Many interactions occur transiently or
under specific conditions and it is therefore
important to study the dynamics of the
interaction within the context of the organism.

¾ The spatially or temporally regulated

interactions can be analyzed with molecular
imaging tools, such as fluorescence resonance
energy transfer (FRET).

Curr Opin Chem Biol. 2001, 5, p60

Example:
Interaction of Gαi3 and calnuc fusion proteins
monitored by live-cell FRET

Colors range between

blue (lowest FRET) and
red and yellow (highest
FRET)

PNAS 2001, 98 ,p14961-14966

29