Abstract
Capacity and diversity are extremely important to the quality of various phage display libraries. In this work, λ phage-based in vitro package was applied to construct a filamentous phage display antibody library so as to enlarge its capacity and introduce more sequence diversity in the final library. In vivo recombination via Cre recombinase/lox sites was also exploited to create VH/VL combination diversity based on multivalent package of λ phage packaging extracts on phagemid DNA concatemers. The library constructed with 10 μg concatenated phagemid DNA and ten vials of λ phage packaging extracts was calculated to contain 1.40×1010 independent clones. Higher capacity can be easily achieved when more materials are consumed. This strategy is somewhat more efficient than prior methods.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alting-Mees MA, Short JM (1993) Polycos vectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts. Gene 137:93–100
Crameri A, Cwirla S, Stemmer WP (1996) Construction and evolution of antibody-phage libraries by DNA shuffling. Nat Med 2(1):100–102
Geoffroy F, Sodoyer R, Aujame L (1994) A new phage display system to construct multicombinatorial libraries of very large antibody repertoires. Gene 151:109–113
Hawkins RE, Russell SJ, Winter G (1992) Selection of phage antibodies by binding affinity. Mimicking affinity maturation. J Mol Biol 226(3):889–896
Hoogenboom HR (2002) Overview of antibody phage-display technology and its applications. Methods Mol Biol 178:1–37
Hoogenboom HR, Winter G (1992) By-passing immunization: human antibodies from synthetic repertoires of germline VH Gene segments rearranged in vitro. J Mol Biol 227:381–388
Hoogenboom HR, Griffiths AD, Johnson KS, Chiswell DJ, Hudson P, Winter G (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19(15):4133–4137
Jackson JR, Sathe G, Rosenberg M, Sweet R (1995) In vitro antibody maturation. Improvement of a high affinity, neutralizing antibody against IL-1 beta. J Immunol 154(7):3310–3319
Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (1991) By-passing immunization: Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222:581–597
McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554
Miwa T, Matsubara K (1982) Identification of sequences necessary for packaging DNA into lambda phage heads. Gene 20:267–279
Perelson AS, Oster GF (1979) Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self–non-self discrimination. J Theor Biol 81:645–670
Sblattero D, Bradbury A (2000) Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnol 18:75–80
Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317
Sternberg N, Hamilton D (1981) Bacteriophage P1 site-specific recombination, I. Recombination between loxP sites. J Mol Biol 150:467–486
Tsurushita N, Fu H, Warren C (1996) Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries. Gene 172:59–63
Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC, McCafferty J, Hodits RA, Wilton J, Johnson KS (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14:309–314
Waterhouse P, Griffiths AD, Johnson KS, Winter G (1993) Combinatorial infection and in vivo recombiantion: a strategy for making large phage antibody repertoires. Nucleic Acids Res 21(9):2265–2266
Willats WG (2002) Phage display: practicalities and prospects. Plant Mol Biol 50(6):837–854
Acknowledgements
We are grateful to Dr. Jianlong Lou for providing vector pDAN5 and strain BS1365. This work was supported by a grant from the National Nature Science Foundation of China (No. 20333010).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Cen, X., Bi, Q. & Zhu, S. Construction of a large phage display antibody library by in vitro package and in vivo recombination. Appl Microbiol Biotechnol 71, 767–772 (2006). https://doi.org/10.1007/s00253-006-0334-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-006-0334-5