Phage Display A Practical Approach

by Clackson, Tim; Lowman, Henry B.

• ISBN13: 9780199638734
• ISBN10: 019963873X
• Edition: 1st
• Format: Paperback
• Copyright: 2004-05-06
• Publisher: Oxford University Press

Summary

Phage display has become established as a powerful protein engineering method for identifying polypeptides with novel properties, and altering the properties of existing ones. Although the technique is widely used in biological research and drug discovery, it remains technically challenging, and new applications and procedures continue to evolve. Phage Display - A Practical Approach is an up-to-date, comprehensive and integrated experimental guide to the technique, useful for novice and expert alike. The book aims to enable researchers to design and undertake all aspects of a phage display project, from designing an experimental strategy and constructing a library to performing selections and analyzing the results. An introductory chapter provides an overview of phage biology and phage display, including guidelines for planning a successful phage display experiment. Individual chapters provide protocols for constructing libraries using oligonucleotide-directed mutagenesis or DNA recombination, performing binding selections, and analyzing the binding activities of selected phage clones. Separate chapters then cover common applications, including selection of ligands from peptide libraries, generation of phage antibody libraries and isolation and optimization of antibodies, selection of DNA binding proteins, and expression cloning using cDNA display. Further chapters describe alternative selection strategies, such as selection using immune sera, selection based on enzymatic activity or protein stability, and selection in vivo. Protocols and chapters are extensively cross-referenced, allowing readers to move beyond the specific examples given to customize the procedures to their own protein or selection system of interest. Written by experts in the field, Phage Display - A Practical Approach provides a comprehensive guide to the design and execution of phage display projects, for all those using the technique in basic research and drug discovery.

Table of Contents

Protocol list
List of abbreviations
List of contributors
Introduction to phage biology and phage display	1	(26)
Marjorie Russel
Henry B. Lowman
Tim Clackson
Introduction	1	(1)
Biology of filamentous phage	2	(8)
Introduction	2	(1)
Structure of the phage particle	2	(3)
Infection	5	(1)
Replication	6	(1)
Genes and gene expression	7	(1)
Physiology of phage assembly	8	(1)
The mechanics of phage assembly	9	(1)
Coat proteins used for display	10	(1)
pVIII	10	(1)
pIII	11	(1)
Starting a phage display project	11	(6)
Feasibility of display	11	(2)
Phage or phagemid vector?	13	(1)
Polyvalent or monovalent display?	14	(1)
Helper phage	15	(1)
General protocols for phage preparation and quantitation	15	(2)
General principles of a phage display project	17	(3)
Making a library	17	(2)
Selection	19	(1)
Analysis of clones	19	(1)
Common problems	20	(1)
Library quality	20	(1)
Expression editing	21	(1)
Over-selection	21	(1)
Alternative display systems	21	(1)
Commercial sources of phage display libraries and kits	22	(5)
References	24	(3)
Constructing phage display libraries by oligonucleotide-directed mutagenesis	27	(16)
Sachdev S. Sidhu
Gregory A. Weiss
Introduction	27	(1)
Considerations for library design	27	(2)
Site-directed mutagenesis	27	(1)
Degenerate codon design	27	(1)
Theoretical versus actual diversity	28	(1)
Oligonucleotide-directed mutagenesis	29	(2)
Oligonucleotide-directed mutagenesis versus cassette mutagenesis	29	(1)
The chemistry and biology of oligonucleotide-directed mutagenesis	29	(2)
Construction of an inactive template	31	(1)
Library construction and storage	31	(8)
Preparation of single-stranded DNA template	32	(2)
In vitro synthesis of heteroduplex CCC-dsDNA	34	(2)
E.coli electroporation and production of library phage	36	(2)
Library storage and reinfection	38	(1)
Biological reagents	39	(4)
References	41	(2)
In vitro DNA recombination	43	(18)
Kentaro Miyazaki
Frances H. Arnold
Introduction	43	(1)
Background to in vitro DNA recombination	43	(2)
Use of in vitro DNA recombination in directed evolution	43	(1)
Applications of in vitro DNA recombination	44	(1)
Recombination statistics	44	(1)
Methods for in vitro DNA recombination	45	(16)
Stemmer method	45	(2)
Random DNA fragmentation with endonuclease V from E. coli	47	(2)
Random priming recombination	49	(1)
Staggered Extension Process (StEP)	50	(2)
In vitro heteroduplex formation and in vivo repair (heteroduplex recombination)	52	(1)
Choice of recombination method	53	(3)
Removal of background	56	(2)
Technical tips	58	(1)
References	59	(2)
Phage selection strategies for improved affinity and specificity of proteins and peptides	61	(24)
Mark S. Dennis
Henry B. Lowman
Introduction	61	(2)
Vector considerations	63	(3)
Monovalent and polyvalent phage display	63	(1)
Confirming display	64	(1)
Protein expression from phagemid vectors	64	(1)
Vector construction and phagemid preparation	64	(2)
Library design	66	(2)
Hard randomization	67	(1)
Soft randomization	68	(1)
Target presentation	68	(3)
Direct immobilization	68	(1)
Solution binding	69	(1)
Blocking	69	(1)
Pilot selection	70	(1)
Selection	71	(8)
Binding buffer considerations	72	(1)
Stringency of selection	72	(1)
Competitive selection	73	(1)
Elution of bound phage	74	(1)
Amplification	75	(2)
Monitoring selection	77	(2)
Screening prior to analysis	79	(1)
DNA sequence analysis	80	(2)
When to sequence	80	(1)
Sequence consensus and sibs	80	(1)
Evaluating the consensus	81	(1)
Evaluation of phage clones	81	(1)
Troubleshooting	82	(3)
References	82	(3)
Rapid screening of phage displayed protein binding affinities by phage ELISA	85	(10)
Warren L. DeLano
Brian C. Cunningham
Introduction	85	(1)
Parameters governing phage binding assays	86	(2)
Performing phage binding assays	88	(6)
Preparation of phage samples	88	(1)
Titration of target immobilization and phage addition	89	(2)
Measurement of phagemid binding to target	91	(1)
Fitting of displacement curves	92	(2)
Concluding remarks	94	(1)
References	94	(1)
Identification of novel ligands for receptors using recombinant peptide libraries	95	(22)
Steven E. Cwirla
Christopher R. Wagstrom
Christian M. Gates
William J. Dower
Peter J. Schatz
Introduction	95	(1)
Description of the peptide display systems	96	(2)
Constructing random peptide libraries in pVIII phagemid	98	(7)
Screening pVIII phagemid libraries	105	(3)
Characterization of recovered clones	108	(2)
Lead optimization strategies	110	(1)
Constructing secondary libraries in the ``headpiece dimer'' system	111	(1)
Affinity selection of headpiece dimer libraries	111	(4)
Transfer to MBP for analysis	115	(1)
Conclusions	115	(2)
References	116	(1)
Substrate phage display	117	(18)
David J. Matthews
Marcus D. Ballinger
Introduction	117	(1)
Preparation of substrate library constructs	118	(3)
Choice of binding pair	120	(1)
Design and construction of substrate cassettes	120	(1)
Substrate selection procedure	121	(4)
Characterization of selected sequences	125	(5)
Initial sequence analysis	125	(1)
``Expression editing'' and other potential complications	125	(1)
Detailed characterization of substrates	126	(4)
Variations and future directions	130	(2)
Substrate subtraction libraries	130	(1)
Substrate specificity of a peptide ligase	130	(1)
Substrate specificity of protein kinases	131	(1)
In vivo selection with retroviral display vectors	131	(1)
Conclusions	132	(3)
References	132	(3)
Protease-based selection of stably folded proteins and protein domains from phage display libraries	135	(20)
Mihriban Tuna
Michael D. Finucane
Natalie G. M. Vlachakis
Derek N. Woolfson
Introduction	135	(1)
The methodology	135	(3)
General procedures and a specific example---rescue of stable ubiquitin variants from a library of hydrophobic core mutants	138	(14)
Construction of a phagemid library of hydrophobic core mutants	139	(6)
Characterization of the phagemid library	145	(1)
Protease selection of stably folded ubiquitin variants	145	(7)
Potential new applications for protease-based selection in phage display	152	(3)
References	153	(2)
Phage display of zinc fingers and other nucleic acid-binding motifs	155	(16)
Mark Isalan
Yen Choo
Introduction	155	(1)
Preliminary considerations in creating a DNA-binding protein phage display library	155	(3)
Is phage display appropriate?	155	(1)
Is the DNA-binding mode of the protein understood?	156	(1)
Which regions of the protein should be randomized?	157	(1)
Polyvalent or monovalent display?	157	(1)
Choice of protein scaffold and selection strategy	158	(1)
Constructing a phage library cassette	158	(3)
Phage vector preparation and library construction	161	(3)
Phage selections	164	(1)
Analysis of selected phage clones	164	(7)
Acknowledgments	168	(1)
References	168	(3)
In vivo and ex vivo selections using phage-displayed libraries	171	(22)
Jason A. Hoffman
Pirjo Laakkonen
Kimmo Porkka
Michele Bernasconi
Erkki Ruoslahti
Introduction	171	(1)
In vivo and ex vivo phage display	171	(1)
Phage vectors	172	(1)
Control comparisons for in vivo and ex vivo phage display	172	(1)
The selection process	173	(8)
Identification of individual phage	181	(10)
PCR and sequencing of the insert coding region	181	(3)
Establishing that individual clones specifically bind organ or tissue vasculature	184	(7)
Concluding remarks	191	(2)
Acknowledgments	191	(1)
References	191	(2)
Screening phage libraries with sera	193	(30)
Paolo Monaci
Riccardo Cortese
Introduction	193	(1)
Construction of phage libraries	193	(1)
Random peptide libraries	193	(1)
Natural epitope libraries	194	(1)
Selection of phage-displayed peptides binding to serum antibodies	194	(7)
Selection strategies	201	(1)
Serial selection	201	(1)
Parallel selection	201	(1)
Screening individual clones	201	(7)
Immuno-screening	201	(4)
DNA-based screening	205	(3)
Characterization of selected clones	208	(8)
ELISA	208	(2)
Characterization of binding specificity by affinity purification	210	(1)
Cross-inhibition assay	211	(3)
DNA sequencing	214	(2)
Epitope maturation	216	(7)
Acknowledgment	220	(1)
References	220	(3)
Interaction cloning using cDNA libraries displayed on phage	223	(20)
Laurent Jespers
Marc Fransen
Introduction	223	(1)
Vectors for display of cDNA libraries on phage	224	(1)
Filamentous bacteriophage	224	(1)
Lytic bacteriophage	224	(1)
Cloning of cDNA libraries in gVI-based display vectors	225	(7)
Description of the pG6 vectors	225	(1)
Preparation of cDNA inserts from pre-made λGT11 libraries by PCR	225	(4)
Ligation and transformation	229	(3)
Evaluation and storage of the gVI-cDNA library	232	(1)
Affinity selection of gVI-cDNA fusion phage	232	(5)
Rescue and purification of the phagemid libraries	232	(2)
Preparation of biotinylated bait	234	(1)
Panning procedure with biotinylated bait	234	(1)
Immunoaffinity panning procedure	234	(3)
Analysis of selected clones	237	(4)
Phage ELISA	238	(2)
Sequence analysis and full-length cloning of the selected cDNAs	240	(1)
Applications	241	(2)
Acknowledgments	242	(1)
References	242	(1)
Phage antibody libraries	243	(46)
Andrew R. M. Bradbury
James D. Marks
Introduction	243	(1)
Phage antibody library construction	243	(23)
The source of V-region diversity	247	(5)
The source of natural V-genes	252	(4)
Assembly and cloning of V-gene repertoires	256	(10)
Preparing phage antibodies for selection on antigen	266	(4)
Selecting antigen-specific antibodies from phage libraries	270	(5)
Identification and characterization of antigen-binding antibodies	275	(8)
Purification of soluble scFv fragments	283	(1)
Troubleshooting	283	(6)
References	286	(3)
Affinity maturation of phage antibodies	289	(28)
Ulrik B. Nielsen
James D. Marks
Introduction	289	(1)
Where and how to diversify the antibody gene sequence	290	(19)
Construction of scFv libraries with diversified CDR3s by site-directed mutagenesis	292	(8)
Construction of chain shuffled libraries for affinity maturation	300	(5)
Construction of light chain shuffled libraries	305	(4)
Selection and screening for higher affinity antibodies	309	(3)
Screening for scFv with improved off-rates	312	(5)
References	314	(3)
Appendix Non-standard suppliers	317	(6)
Index	323