噬菌体展示

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