Welcome to the pComb 3 page, home of the Worlds First Antibody Fab Fragments Displayed on Phage. If you would like to receive training in phage display of antibody and peptide libraries, I teach a Cold Spring Harbor Laboratory course on the subject every fall. The deadline for applications is typically July and the course is given in November. For more information, see the Cold Spring Harbor web site at www.cshl.edu.
Best of luck in your research,
C.F. Barbas III
-Development of the
first phage display systems for the display of large proteins, particularly
50 KD heterodimeric antibody Fab fragments
-Development of the first phage displayed Human Antibody Libraries
-Cloning of the first High Affinity Human Monoclonal Antibodies using Phage Display
-Development of Methodologies for the construction and selection of phage display libraries of Human, Mouse, Rabbit and Chicken antibody Fabs and scFvs
-Development of the first Synthetic Antibody Libraries
-Development of the first Zinc Finger Display Libraries
-Development of the first Motif Grafted Antibodies
-Development of one the most potent HIV-1 neutralizing antibodies known, IgG-b12
-Development of robust methods to improve Antibody affinity- CDR Walking
-Development of the first in vitro evolved Antibodies with enhanced biological activity, the anti-HIV-1 antibody 3B3
-Development of phage-based methods for the humanization of mouse and rabbit Antibodies
-Development of a wide range of anti-Cancer Antibodies
-Development of novel Catalytic Antibodies and Catalytic peptides
The pComb 3 vectors, related vectors for phage display and protein expression are available upon request. Vectors designed in our lab for phage display and construction of antibody, protein, and peptide libraries can be requested by downloading a TSRI MTA (Material Transfer Agreement) Request form, filling out the sections Material Requested and Requestor with PIs information, then emailing to firstname.lastname@example.org or faxing to 858-784-2583. Processing of the request is done by the TSRI Office of Technology Development. There is a fee for processing and shipping of these plasmids. See links below for descriptions and downloadable maps and sequences. Details about our plasmids and protocols can be found in:
Barbas, C. F., III; Burton, D. R.; Scott, J.K., Silverman, G.J. Eds. (2001) Phage Display: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 736 pages. Information about the manual can be found by clicking on the title or by purchasing directly from www.cshlpress.com or www.Amazon.com.
pComb3X is the newest of the pComb vectors. Improvements over pComb3 include increased stability and introduction of SfiI cassette for cloning of full Fab, scFv, peptide and other protein for phage display. 6xHis and HA tags allow for purification and detection. An amber stop codon was introduced to turn-off expression of the pIII fusion protein by switching to a non-supressor strain of E. coli allowing production of soluble protein without subcloning. Alternatively, the gene for phage protein pIII can be removed by SpeI/NheI digest. pComb3XSS is the vector we normally ship. The SS refers to the double stuffer, a 1200bp stuffer in the Fab light chain cloning region bounded by SacI and XbaI restriction sites and a 300bp stuffer in Fab heavy chain cloning region bound by XhoI and SpeI restriction sites. Also, the 1600bp double stuffer (both stuffer plus the leader sequence between the Fab light chain and heavy chain cloning regions) can be removed by SfiI digest so that non-Fab genes of interest can also by cloned. pComb3XTT and pComb3XLambda are only needed for the construction of chimeric Fab libraries as described in Phage Display: A Laboratory Manual. The TT refers to the human Fab to tetanus toxin from which the kappa light chain constant region and Fd can be amplified. pComb3XLambda contains a non-functional Fab from which the lambda light chain constant region can be amplified.
Andris-Widhopf, J.; Rader, C.; Steinberger, P.; Fuller, R.; Barbas III, C.F. (2000) Methods for the Preparation of Chicken Monoclonal Antibody Fragments by Phage Display. J. Immun. Meth., 242, 159-181.
C; Popkov, M.; Neves, J.A.; Barbas III, C.F. (2002) Integrin _v_3 Targeted Therapy
of Kaposis Sarcoma with an In Vitro Evolved Antibody. FASEB, 16(14):2000-2.
Berry, J.D.; Rutherford, J.; Silverman, G.J.; Kaul, R.; Elia, M.; Gobuty, S.; Fuller, R.; Plummer, F.A.; & Barbas III, C.F. (2003) Development of Functional Human Monoclonal Single-Chain Variable Fragment Antibody Against HIV-1 From Human Cervical B cells. Hybridoma and Hybridomics, 22(2):, 97-108.
N, Popkov M, Beerli RR, Chung J, McGavern DB, Rader C, Barbas CF 3rd. (2003)
Intra-diabodies: Bispecific, tetravalent antibodies for the simultaneous functional
knockout of two cell surface receptors. J Biol Chem., 278(48):, 47812-9.
P.; Sutton, J.K.; Rader, C.; Elia, M.; and Barbas III, C. F. (2000) Generation
and Characterization of a Recombinant Human CCR5-specific Antibody: A Phage
Display Approach for Rabbit Antibody Humanization. J. Biol. Chem., 275,,
J.; Kilva, F.; Freitas-Vieira, A.; Santa-Marta, M.; Malho, R.; Yang, X.; Gabuzda,
D.; and Barbas III, C.F. (2002) Functional Neutralization of HIV-1 Vif Protein
by Intracellular Immunization Inhibits Reverse Transcription and Viral Replication.
J. Biol. Chem., 277(35):32036-45.
M.; Mage, R.G.; Alexander, C.B.; Thundivalappil, S.; Barbas III, C.F.; Rader,
C. (2003) Rabbit immune repertoires as sources for therapeutic monoclonal antibodies:
The impact of Kappa Allotype-correlated variation in cysteine content on antibody
libraries selected by phage display. J. Mol. Biol., 325:325-335.
J.; Rader, C.; Popkov, M.; Hur, Y.-M.; Kim, H.-K.; Lee, Y.-J.; & Barbas
III, C. F. (2004) Integrin _IIb_3 specific synthetic human monoclonal antibodies
and HCDR3 peptides that potently inhibit platelet aggregation, FASEB
M.; Jendreyko, N.; Gonzalez-Sapienza, G.; Mage, R.G.; Rader, C.; Barbas III,
C.F. (2004) Human/mouse cross-reactive anti-VEGF receptor 2 recombinant antibodies
selected from animmune b9 allotype rabbit antibody library, J. Immunol. Methods,
M.; Rader, C.; Barbas III, C.F. (2004) Isolation of human prostate cancer reactive
antibodies using phage display technology, J. Immunol. Methods, 291:137-151.
Tanaka, F.; Fuller, R.; Barbas III, C.F. (2005) Development of Small Designer Aldolase Enzymes: Catalytic Activity, Folding, and Substrate Specificity. Biochemistry, 44:7583-7592.
Family (Genebank AF268280) Maps
pMalCSS (cytoplasmic expression) Text
pMalPSS (periplasmic expression) Text
pComb3H is the second generation of pComb vector. Improvements over pComb3 include increased stability and introduction of a new cloning region utilizing SfiI restriction sites for cloning of full Fab, scFv, peptide and other proteins for phage display. pComb3HSS contains the same double stuffer or SS as described above for pComb3XSS. pComb3HTT contains a human Fab to tetanus toxin which can be used as a control for Fab expression. Soluble Fab can be expressed by removing the gene for the pIII phage fusion protein by SpeI/NheI digest. Sufficient soluble protein is also found in the periplasmic space as a result of proteolysis. For expression of non-Fab proteins, subcloning to an expression vector with tags for detection may be necessary. We can provide arabinose inducible pAraH6HATT which contains the human Fab to tetanus toxin and a compatible SfiI cloning region as well as a similarly equipped Maltose- binding protein fusion vector pMal-SS (either cytoplasmic or periplasmic). Our version of the pMal vectors, originally obtained from New England Biolabs, are modified with the SfiI cloning cassette and SS double stuffer for easy transfer from pComb3H.
S.P.; Lee, J.; Wagenknecht, R.; Silverman, G.J. (2000) Characterization of Superantigen-Induced
Clonal Deletion with a Novel Clan III-Restricted Avian Monoclonal Antibody:
Exploiting Evolutionary Distance to Create Antibodies Specific for a Conserved
VH Region Surface. J. Immun., 164, 4730-4741.
F., Barbas III, C.F. (2001) Phage display selection of peptides possessing aldolase
activity. J. Chem. Soc., Chem. Commun. 8:769-770.
C.; Cheresh, D.; Barbas III, C.F. (1998) Phage display approach for rapid antibody
humanization: Designed combinatorial V gene libraries. Proc. Natl. Acad.
Sci. USA, 95, 8910-8915.
P.; Sutton, J.K.; Rader, C.; Elia, M.; and Barbas III, C. F. (2000) Generation
and Characterization of a Recombinant Human CCR5-specific Antibody: A Phage
Display Approach for Rabbit Antibody Humanization, J. Biol. Chem., 275,36073-36078.
C. and Barbas III, C.F. (1997) Phage Display of Combinatorial Antibody Libraries.
Current Opinion in Biotechnology 8, 503-508.
C.; Ritter, G.; Nathan, S.; Elia, M.; Gout, I.; Jungbluth, A.A.; Cohen, L.S.;
Welt, S.; Old, L.J.; Barbas III, C.F. (2000) The rabbit antibody repertoire
as a novel source for the generation of therapeutic human antibodies. J.
Biol. Chem., 275, 13669-13676.
D.J.; Dreier, B.; Beerli, R.R..; Barbas III, C.F. (1999) Towards controlling
gene expression at will: Selection and design of zinc finger domains recognizing
each of the 5 - GNN-3 DNA target sequences. Proc. Natl. Acad.
Sci, USA, 96, 2758-2763.
H., Yang, W.-P., and Barbas III, C.F. (1995) Building Zinc fingers by selection:
Towards a therapeutic application. Proc. Natl. Acad. Sci. USA, 92:344-348.
Yang, W.-P., Green, K., Pinz-Sweeney, S., Briones, A.T., Burton, D.R., and Barbas III, C.F. (1995) CDR Walking Mutagenesis for the Affinity Maturation of a Potent Human anti-HIV-1 Antibody into the Picomolar Range. J. Mol. Biol. 254:392-403.
The Original pComb
Text & Map
pComb8 Text & Map
The original pComb3 vector
was designed for phage display of Fabs which are cloned in one chain at a time
using SacI/XbaI restriction sites (for the light chain) and XhoI/SpeI restriction
sites (for the heavy chain). This vector system has also been used for the display
of a wide variety of other proteins like zinc fingers, peptides, and cDNA fragments.
Phage can be produced which express Fab or other proteins or peptides of interest
fused to the phage pIII protein for expression on the head of the phage. Soluble
Fab can be expressed by removing the gene for the pIII phage fusion protein
by SpeI/NheI digest. Sufficient soluble protein is also found in the periplasmic
space as a result of proteolysis. pComb8 is nearly identical but contains the
phage pVIII fusion protein for multi-valent expression along the sides of the
phage via fusion with pVIII.
Barbas III, C.F. (1995)
Synthetic Human Antibodies. Nature Medicine 1,837-839.
Barbas III, C.F., Bjorling,
E., Chiodi, F., Dunlop, N., Cababa, D., Jones, T.M., Zebedee, S.L., Persson,
M.A.A., Nara, P.L., Norrby, E. and Burton, D.R. (1992) Recombinant Human Fab
fragments neutralize human type 1 immunodeficiency virus in vitro. Proc.
Natl. Acad. Sci. USA 89, 9339-9343
Barbas III, C.F., Kang,
A.S., Lerner, R.A. and Benkovic, S.J. (1991) Assembly of combinatorial antibody
libraries on phage surfaces: The gene III site. Proc. Natl. Acad. Sci. USA
Barbas III, C.F., Crowe,
J.E., Cababa, D., Jones, T.M., Zebedee, S.L., Murphy, B.R., Chanock, R.M. and
Burton, D.R. (1992) Human Monoclonal Fab Fragments derived from a combinatorial
library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity.
Proc. Natl. Acad. Sci. USA 89, 10164-10168
Barbas III, C.F., Persson, M.A.A., Koenig, S., Chanock, R.M., Burton, D.R. and Lerner, R.A. (1992) A large array of human monoclonal antibodies to HIV-1 from combinatorial libraries of an asymptomatic seropositive individual. Vaccines '92: Modern Approaches to New Vaccines including Prevention of AIDS, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York , 9-12
Barbas III, C.F., Bain,
J.D., Hoekstra, D.M. and Lerner, R.A. (1992) Semi-synthetic combinatorial antibody
libraries: A chemical solution to the diversity problem. Proc. Natl. Acad.
Sci. USA, 89:4457-4461.
Lerner, R.A., Kang, A.S.,
Bain, J.D., Burton, D.R., Barbas III, C.F. (1992) Antibodies without immunization.
Barbas III, C.F., Amberg,
W., Somincsits, A., Jones, T.M. and Lerner, R.A. (1993) Selection of human anti-hapten
antibodies from semisynthetic libraries. Gene, 137:57-62.
Burton, D.R., Barbas III,
C.F., Persson, M.A.A., Koenig, S., Chanock, R.M. and Lerner, R.A. (1991) A large
array of human monoclonal antibodies to type 1 human immunodeficiency virus
from combinatorial libraries of asymptomatic seropositive individuals. Proc.
Natl. Acad. Sci. USA 88, 10134-10137
Barbas III, C.F., Languino,
L.R. and Smith, J.W. (1993) High Affinity Self-Reactive Human Antibodies by
Design and Selection: Targeting the Integrin Ligand Binding Site, Proc. Natl.
Acad. Sci. USA 90:10003-10007.
Smith, J.W., Hu, D., Satterthwait,
A.C., Pinz-Sweeney, S. and Barbas III, C.F. (1994) Building Synthetic Antibodies
as Adhesive Ligands for Integrins. J. Biol. Chem. 269:32788-32795.
Barbas III, C.F., and Wagner, J. (1995) Synthetic Human Antibodies: Selecting and Evolving Functional Proteins. Methods, A Companion to Methods in Enzymology 8, 94-103.
Other Related VectorspJF3H Text & Map
Modified version of pComb3H
containing Jun, Fos and a cloning site for cDNA expression. Developed in the
lab of Gregg Silverman at UCSD.
Barbas, C. F., III; Burton,
D. R.; Scott, J.K., Silverman, G.J. Eds. (2001) Phage Display: A Laboratory
Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, , 736 pages.
pIgG Text &
Vector for conversion of
Fab to IgG for expression in mammalian cells.
Rader, C; Popkov, M.; Neves, J.A.; Barbas III, C.F. (2002) Integrin avb3 Targeted Therapy of Kaposis Sarcoma with an In Vitro Evolved Antibody . FASEB, 16(14), 2000-2002.
Now Available at www.cshlpress.com* or www.Amazon.com:
Phage Display: A Laboratory
By Carlos F. Barbas III, The Scripps Research Institute;
Dennis R. Burton, The Scripps Research Institute;
Jamie K. Scott, Simon Fraser University;
Gregg J. Silverman, University of California, San Diego
2001, 736 pp., illus., index
Paperback $75 ISBN 0-87969-546-3
*Customers in Europe, the Middle East, and Africa should check for this publication at the CSHL Press Europe website.
technology has begun to make critical contributions to the study of molecular
DNA sequences are cloned into phage, which then present on their surface the proteins encoded by the DNA.
Individual phage are rescued through interaction of the displayed protein with a ligand, and the specific phage
is amplified by infection of bacteria.
technology is powerful but challenging and the aim of this manual is to provide
instruction in its theoretical and applied so that any scientist with even modest molecular biology experience
can effectively employ it. The manual reflects nearly a decade of experience with students of greatly varying
technical expertise andexperience who attended a course on the technology at Cold Spring Harbor
technology is growing in importance and power. This manual is an unrivalled
expertise in its execution and application.
SECTION 1 PHAGE DISPLAY
Chapter 1. Filamentous Phage Biology
Chapter 2. Phage-display Vectors
Chapter 3. Antibody Libraries
Chapter 4. Peptide Libraries
Chapter 5. Functional Domains and Scaffolds
Chapter 6. Gene Fragment Libraries and Genomic and cDNA Expression Cloning
SECTION 2 ANTIBODY LIBRARIES
Chapter 7. Overview: Amplification of Antibody Genes
Chapter 8. Generation of Antibody Libraries: Immunization, RNA Preparation, and cDNA Synthesis
Chapter 9. Generation of Antibody Libraries: PCR Amplification and Assembly of Light- And
Heavy-chain Coding Sequences
Chapter 10. Selection from Antibody Libraries
Chapter 11. Analysis of Selected Antibodies
Chapter 12. Production and Purification of Fab and scFv
Chapter 13. Antibody Engineering
SECTION 3 PEPTIDE LIBRARIES
Chapter 14. Overview: Peptide Libraries
Chapter 15. General Phage Methods
Chapter 16. Production of Peptide Libraries
Chapter 17. Screening Peptide Libraries
Chapter 18. Analysis of Phage-borne Peptides
Chapter 19. Construction and Use of pIII-displayed Peptide Libraries
SECTION 4 OTHER LIBRARIES AND OTHER METHODS OF PANNING
Chapter 20. Construction and Selection from Gene Fragment Phage-display Expression Libraries
Chapter 21. Construction and Selection from cDNA Phage-display Expression Libraries
Chapter 22. In Vivo Selection of Phage-display Libraries
Chapter 23. Cell-surface Selection and Analysis of Monoclonal Antibodies from Phage Libraries
SECTION 5 APPENDICES
Appendix 1. Useful Information
Appendix 2. Recipes
Appendix 3. General Procedures
Appendix 4. Cautions
Appendix 5. Suppliers
Appendix 6. Trademarks