Phage Display Technology
The development of monoclonal antibody (mAb) technology (1 ) has had a significant impact on many fields of research, in particular immunology. However, the method has limitations. The use of recombinant DNA technology and demonstration by Smith 1985 (2 )
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4 Phage Display Technology Michael Johns 1. Introduction The development of monoclonal antibody (mAb) technology (1) has had a significant impact on many fields of research, in particular immunology. However, the method has limitations. The use of recombinant DNA technology and demonstration by Smith 1985 (2) that peptides can be expressed on the surface of filamentous bacteriophages have permitted the development of a powerful new methodology for the generation and isolation of novel antibody-based reagents for both research and clinical application. Filamentous bacteriophage (e.g., M13) have a single-stranded DNA genome encapsulated by multiple copies of the major coat protein pVIII (Fig. 1). The number of copies of pVIII is variable, thus allowing additional DNA to be incorporated and packaged within the virion without any adverse effects (3). The gene III product, expressed at one end of the bacteriophage, is particularly important in the life cycle of the bacteriophage; it is responsible for infecting male Fv-positive bacteria (e.g., Escherichia coli strain TG1), transfer of DNA to the bacteria, and initiation of replication (4). In the context of phage display technology it is also the most common (but not only) site for the expression of recombinant proteins, such as sFv (Fig. 1). It is possible to express sFv on all copies of the gene III product (5); however, this is the exception rather than the rule. A variety of peptides and proteins have been expressed on the surface of filamentous phage, including fragments of EcoRI endonuclease, IL-3, human growth hormone, alkaline phosphatase, and antibody fragments (2,6–8). As a prerequisite to expressing antibody fragments on the surface of bacteriophage, the heavy and light variable region genes for immunoglobulin must be cloned.
From: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
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Fig. 1. Diagrammatic representation of a filamentous phage expressing sFv on the gene III product. 2700 copies of the major coat protein (pVIII) encapsulate a singlestranded DNA genome. Five copies of the gene III product cap one end of the phage and are the principal site for expression of recombinant proteins. pVI, pVII, and pIX constitute the other minor coat proteins. Filamentous bacteriophages are approx 1 μM in length and 6–7 nm in diameter.
Typically this is done by polymerase chain reaction (PCR) (see Chapter 36) and can be used to clone either specific antibody fragments or families of antibody genes. Cloning families of antibody genes has led to the development of libraries of antibody fragments expressed on the surface of bacteriophage. This chapter is concerned with phage display in the context of antibody engineering and the application of antibody fragments (sFv and Fab) selected from diverse libraries. 2. Phage Display Libraries A phage lambda system was used originally to screen diverse combinatorial libraries of Fab fragments developed from im
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