1
1
cDNA Display Libraries in T7 Based Vectors
T7SelectTM Phage Display System
Antibody/Antigen • Receptor/Ligand • Enzyme/Substrate
2
Tools:
cDNA libraries
Restriction Mapping
SNP discovery
DNA sequence analysis
I. Gene DiscoveryI. Gene Discovery
II. Functional AnalysisII. Functional Analysis
Protein
“Proteomics”
Tools:
Mass Spectrometry
Two- Hybrid Systems
Viral Vectors
siRNA
Mutagenesis
Phage display
2D
pET system
RNA
Tools:
Microarrays (Qualitative)
QPCR (Quantitative)
3
Phage Display System
M13 Phagedisplay technology developed by George
Smith and coworkers in 1985
• Identity protein-protein interaction
•Capture and isolate ligand, substrate, inhibitor/ activator, episode, etc.
•Modify protein and improve the affinity binding ability
•Medicine development
4
Phage Display
Lead Hv Link Lv E PIII
融合蛋白
PIII
Genotype Phenotype
5
Conventional library
screening
6
Biopanning
Bind
Wash
Titer
Elute
Solid
Support
“Bait”
2
7
l immobilize target in
microtiterwells
ladd phage to
microtiterwells
lwash unbound phage
from wells
lelute bound phage
lrepeat panning
l plate out library
lawait plaque/
colony development
lapply nitrocellulose filter
lblock
l1° antibody, wash
l2° antibody conjugate, wash
lvisualize
lline up plate with filter
lpick plaque
lelute
lreplate
Biopanning Screening
8
Display Systems
l M13
l T7
l l
l E. coli
lYeast
l gp64 fusions in insect cells
9
Comparison of bacteriophages
l filamentous
l ss
l 6.3kb
l circular
l release by
secretion
l icosahedron
l ds linear
l 50kb
l 12nt cohesive ends
l temperate
l release by lysis
l icosahedron
l ds linear
l 40kb
l 160bp redundant
l virulent
l release by lysis
M13 l T7
10
M13 life cycle
Assembly site
pI
pIV
pXI
pV
DNA (+)
pVIII
pVIII
pVII, pIX
F pilus
pVI
pIII
pIII pVI
11
T7SelectTM Phage Display System
a novel gene discovery tool based on the unique properties of bacteriophageT7 (rather than the traditional M13). Target sequences are fused (C-terminal) with the T7 gene 10major capsidprotein so that expressed peptides and proteins are exposed (or“displayed”) on the surface of the virus (T7 bacteriophage).
12
T7 life cycle
3
13
Requirements for Display Vector
•Means to express protein on outside
•Flexible site to accommodate fusion protein
•Means to deliver recombinant DNA
•Resistant to reagents used to wash/elute
14
T7 Virus Structure
15
Bacteriophage T7
•Capsidis made of 415 copies of the major capsid
protein (gene 10 protein)
•Capsid may be assembled from any combination of
2 versions of gene 10 protein
•Genome is linear ds DNA; completely sequenced
•Assembly pathway well studied: in vitro packaging
system developed
16
Bacteriophage T7 genome
17
T7Select® cloning sites
415 copies
< 1 copy
10 copies
18
•1200aa display capacity
•Phage released by lysis
•C-terminal fusions
•Resistant to harsh conditions
Advantages of T7 for displaying
cDNA libraries
4
19
Multiple cloning regions
* *
* *
20
T7Select® vector features
Vector Use Display Number Display Limit Host
T7Select415-1 peptides 415 50 aa BL21
T7Select1-1 peptides 0 -1 1200 aa BLT5403
or proteins BLT5615
T7Select1-2 peptides 0 -1 900 aa BLT5403
or proteins BLT5615
T7Select10-3 peptides 5 -15 1200 aa BLT5403
or proteins BLT5615
21
Genotypes of host strains
BL21: F–ompT hsdSB (rB–mB–) gal dcm
BLT5403: F–ompT hsdSB (rB–mB–) gal
dcm pAR5403 (ampR)
BLT5615: F–ompT hsdSB (rB–mB–) gal dcm
pAR5615 (ampR)
BLT5615rna-Deletion of Rnase I
Other Strains:Origami™ B 5615, Rosetta™ 5615
and Rosetta-gami™ B 5615
22
Analysis of copy number displayed
on T7Select® vectors
Ten-fold dilutions of equivalent amounts of T7Select™415 and T7Select1 phage encoding an HSV epitope were analyzed by Western blotting using the
HSV-Tag™ monoclonal antibody.
23
Target protein accessibility
Units of thrombin incubated with nonrecombinantT7Select™415 phage or T7Select415 displaying the thrombin recognition peptide.
24
•Use high quality mRNA
•Block background binding
•Optimize wash conditions
•Monitor enrichment;rounds of biopanning
(1st:103-105)
Keys to successful phage display of cDNA
5
25
cDNA
library construction
I
poly(A)+
RNA
cDNA
synthesis
end modification
26
cDNA
library construction
II
size fractionate
ligate to vector
package
infect host to express fusions
T7 Packaging Extracts for in
vitro packaging.
High efficience: 2 x 109 pfu /µg
27
OrientExpressTM cDNA synthesis
28
Directional Linker
5' GCTTGAATTCAAGC
29
OrientExpress™ cDNA synthesis
5’ 3’
cDNA is made withmethylated dCTPto protect internal sites
30
cDNA Clone Analysis
M M 1 2 3 4 M
4361 -
2000 -
1500 -
1000 -
750 -
500 -
300 -
150 -
50 -
Lane cDNA Primer mg/ mg RNA
1 oligo( dT) 0.5
2 HindIII RP* 2.5
3 HindIII RP 0.25
4 HindIII RP 0.025
M size markers
* RP = random primers
4mg of a 3256 RNA template was used for cDNA synthesis with the indicated primers and a portion of the cDNAwas analyzed by agarosegel electrophoresis.
6
31
Protection of Internal Hind III Site
in cDNA with 5-me dCTP Incorporation
1 2 3 4
1 unmethylated
2 hemi-methylated
3 fully methylated
4 control
1605 -
1198 -
676 -
350 -
222 -
517 -
cDNAwas synthesized with no methyl dCTP, or adding the methylateddNTPto only the first strand synthesis or in both strands. The resulting cDNAswere digested with Hind III.
32
OrientExpress with T7 Select
Day 1. Make first and second strand cDNA
Blunt cDNA ends for cloning
Ligate to linkers
Day 2. Digest linkers
Size fractionate cDNA
(Cut and prepare vector arms, if necessary)
Ligate cDNA to vector arms
Day 3. Package phage in vitro
Plate phage & pick plaques 3+ hours later
Amplify finished library
Total time: Approximately 3 days
?
Next: Screen library
33
Number of Clones to Screen?
With P=99% probability,
N= 170,000
N = ln(1-P)/ln(1-f)
Peptide/Protein Probe:
10,000 - 30,000 unique mRNA
30% low abundance (<14 copies per cell)
X 3 directional = 5 X 105
X 6 non-directional = 1 X 106
34
Biopanning with T7Select cDNA clones:
Day 1. Immobilize biopanning ligand
(eg., on a 96 -well ELISA plate)
Perform 1st biopanning (Incubate phage lysate
with ligand, wash with TBST, elute bound phage)
Amplify eluted phage (1 -3 hour culture)
Perform 2nd round ofbiopanning
Amplify eluted phage
Day 2. 3 rd round of biopanning if desired
Amplify phage
4 th round of biopanning if desired
Total time: Approximately 2 days
35
•Solid support: plates, affinity resins and magnetic particles
•Wash/elute reagents: 5M NaCl, 4M urea, 2M
guanadine-HCl, 10mM EDTA, 1% SDS, 100mM DTT,
pH 4-10, glutathione
•Binding conditions: Fast on rates select tight
binders, slower on rates permits more clones to bind
Biopanning Conditions
36
cDNA display libraries in T7 based vectors
downstream analysis
Now you have the clone(s) that you selected. Can analyze
by:
•Plaque lift
•Amplify or clone
•PCR analysis/seq.
Stab a plaque and elute in EDTA (usually enough DNA) or
Large quantities of DNA can be purified
7
37
Anal. Biochem. 268: 363 (1999) Used high copy number T7 display vector
to determine mAb specificity.
Mol. Therap . 2: 131 (2000) Demonstrated displayed peptides are
recognized by natural antibodies and induce complement activation.
J. Immunol. Meth. 257: 117 (2001) Used T7 particles displaying specific
peptides as targets for isolation of M13 scFvs.
Literature citations: peptide libraries
38
Biochem. Biophys. Res. Com. 255: 194 (1999) Constructed cDNA library in low
copy number T7 display vector, isolated clones expressing protei ns which bind
to carbohydrates.
EMBO Jour. 18: 1982 (1999) Constructed cDNA library, isolated Ran -binding
domain.
Chem. and Biol. 6: 707 (1999) Human brain cDNA library in mid- copy vector was
used to isolate full length clone which binds to FK506-binding protein.
J. Virol. 73: 7761 (1999) Cat cDNA library was source of phage displaying
proteins which bind feline panleukemia virus.
Nature 405: 85 (2000) cDNA library used to select for clone encoding protein
which recognizes phosphotidylserine on apoptotic cells.
PNAS 98: 12954 (2001) Selective isolation of RNA-binding proteins from cDNA
library.
Literature citations: cDNA libraries
39
Literature citations: cDNA libraries
J. Immunol. 2001, 167: 6009. Biopanned against IgG extracted from brain of
subacute sclerosing panencephalitis patient. Positive clones contained
fragments of nucleocapsid protein of measles virus.
Chem Biol. 2002, 9: 157. Human liver cDNA T7 display library screened against
immobilized doxorubicin. Identified C-terminal domain of nucleolar
phosphoprotein.
40
cDNA display libraries in T7 based vectors
Find One in a BillionFind One in a Billion
本文档为【噬菌体展示】,请使用软件OFFICE或WPS软件打开。作品中的文字与图均可以修改和编辑,
图片更改请在作品中右键图片并更换,文字修改请直接点击文字进行修改,也可以新增和删除文档中的内容。
该文档来自用户分享,如有侵权行为请发邮件ishare@vip.sina.com联系网站客服,我们会及时删除。
[版权声明] 本站所有资料为用户分享产生,若发现您的权利被侵害,请联系客服邮件isharekefu@iask.cn,我们尽快处理。
本作品所展示的图片、画像、字体、音乐的版权可能需版权方额外授权,请谨慎使用。
网站提供的党政主题相关内容(国旗、国徽、党徽..)目的在于配合国家政策宣传,仅限个人学习分享使用,禁止用于任何广告和商用目的。