Research Article: Phage Display on the Base of Filamentous Bacteriophages: Application for Recombinant Antibodies Selection

Date Published: October , 2009

Publisher: A.I. Gordeyev

Author(s): N.V. Tikunova, V.V. Morozova.

http://

Abstract

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

Partial Text

In the mid-eighties, a novel molecular-biological methodology which revolutionized the engineering of peptides and proteins was developed. This approach is known as phage display. It is based on the experiments of George Smith performed in the mid-80s [1]. Initially, Smith demonstrated that an exogenous protein can be expressed on the surface of the filamentous M13 phage. This was achieved by inserting the gene that encoded a part of the EcoRI endonuclease into the ORF of the phage’s minor capsid protein pIII. Using polyclonal antibodies specific to the EcoRI endonuclease, Smith demonstrated the ability of phages carrying the chimeric EcoRI-pIII protein to specifically bind the appropriate antibodies. Furthermore, it was shown that phages with this insertion could be selected from a mixture containing wild-type phages by affine enrichment using polyclonal antibodies against the EcoRI endonuclease.

Phage display methodology and its success are defined by the features of filamentous phages. Currently, several filamentous bacteriophages are known to infect gram-negative bacteria. The best characterized are the M13, f1, and fd phages, which infect Escherichia coli strains that carry an F-conjugative plasmid. The genomes of these phages have been sequenced and are 98 % homologous [6, 7]. Based on this homology and also on the dependence of infection on the presence of an F-plasmid, these phages are all termed Ff-phages.

A number of novel vector molecules have been constructed on the base of filamentous phage DNA. These so-called “phagemids” combine the characteristics of plasmids and phages [10]. Phagemids contain a replication origin and the packaging signal of the Ff-phage, as well as a replication origin for the chosen plasmid, gene III, a polylinker, and an antibiotic-resistance gene [11].

In order to select for target antibodies with the desired characteristics, two important steps are required: first, an adequate phage antibody library should be used; and second, the right biopanning strategy should be chosen.

Different sources of gene repertoire are used for the construction of phage display antibody libraries, depending on the library type. Naive and immune libraries are constructed using naturally reorganized genes, which encode the variable immunoglobulin domains of healthy donors or donors immunized with a certain antigen, respectively (Fig.4). mRNA from the antibody-producing lymphoid cell line is isolated for this purpose: peripheral blood lymphocytes are mainly used, but splenocytes [23, 24], tonsil cells, and B-lymphocytes from the bone marrow, have been used as well [25, 26]. A preliminary in vitro immunization of peripheral lymphocytes can also be used to construct an immune antibody library [27, 28].

The next important step after constructing a library or choosing an available one is the enrichment of the original antibody repertoire with the antibodies specific to a target antigen (Fig.5). This procedure is called “biopanning” or affinity enrichment. Below, we present several biopanning strategies.

Many researchers have faced a number of problems while constructing and using libraries based on filamentous phages. For instance, the low concentration of the original antigen-specific lymphocytes in the population can be a problem while constructing immune libraries. This and similar problems can be avoided by preliminary enrichment of the B-lymphocyte population, using antigen molecules conjugated with magnetic beads [91, 92].

This work was supported by RFBR grant 07-04-12100-ofi, 09-04-01546-a, 07-04-92168-NCNI_a, NATO SFPP 982833, and the “Fundamental science for medicine – 2008” program of the presidium of the Russian Academy of Sciences.

 

Source:

http://