Elsevier

Methods

Volume 32, Issue 3, March 2004, Pages 212-218
Methods

Cloning allergens via phage display

https://doi.org/10.1016/j.ymeth.2003.08.003Get rights and content

Abstract

Although an impressive list of allergenic structures has been elucidated during the last decade by classical cloning methods, the size of the repertoire of molecular structures able to elicit allergic reactions is still unknown. Selective enrichment of cDNA libraries displayed on phage surface with serum IgE from allergic individuals combined with robotic-based high-throughput screening technology has proved to be extremely successful for the rapid isolation of allergens. The basic concept of linking the phenotype, expressed as gene product displayed on the phage coat, to its genetic information integrated into the phage genome, creates fusion proteins covalently associated with the infectious particle itself. Therefore, cDNA libraries displayed on phage surface can be screened for the presence of specific clones using the discriminative power of affinity purification. The selection of IgE-binding clones involves the enrichment of phage binding to serum IgE immobilised to a solid phase during consecutive rounds of affinity selection. As a consequence of the physical linkage between genotype and phenotype, sequencing of the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of the surface-displayed allergen. In spite of some biological limitations imposed by Escherichia coli as expression host, phage surface display technology has strongly contributed to the rapid isolation of a vast variety of IgE-binding structures.

Introduction

A common hallmark of allergic diseases is the production of allergen-specific IgE raised against normally innocuous environmental antigens [1], [2]. In sensitised atopic individuals, allergen exposure induces cross-linking of high-affinity FcεRI receptor-bound IgE on effector cells and, thus, immediate release of anaphylactogenic mediators [2]. Although the mechanisms leading to allergic reactions are quite well understood [3], [4], our knowledge about the repertoire of molecular structures involved in the pathogenesis of allergic reactions is still fragmentary. This is mainly due to the complexity of most of the allergenic sources [5] and to the ability of the immune system of atopic individuals to mount IgE immune responses to a wide variety of different molecular structures. Prominent examples of extremely complex allergenic sources are Aspergillus fumigatus (A. fumigatus) and Dermatophagoides pteronyssinus (house dust mite) reported to contain 81 and 16 IgE-binding proteins, respectively [6], [7]. Although the incidence of sensitisation against a given allergenic structure can vary considerably, also allergens with a low prevalence of sensitisation might be clinically relevant as cross-reactive structures. This is best exemplified by profilin, a minor allergen first cloned from birch [8] and later demonstrated to be an important allergen in many sources [9], [10], and ribosomal P2 protein of A. fumigatus shown to cross-react with the corresponding human protein [11]. After the first report of a cloned allergen in 1988 [12], many allergens have been cloned, characterised, and produced as recombinant proteins. Sequence information and references can be accessed through a database inventoried by the Allergen Nomenclature Sub-Committee of the International Union of Immunological Societies (www.allergen.org). To maintain the integrity of the nomenclature system, only allergens that meet one of the two following criteria are included in this list: (I) A demonstrated prevalence of IgE reactivity above 5% or (II) A minimum of five patients showing IgE reactivity, regardless of the number of patients investigated [13]. However, a distinction between an allergen as defined molecular structure and allergen extracts, though often called allergens in clinical practice, should be made. The term allergen should only be used for single, pure proteins which are able to bind IgE in vitro and to elicit immediate type skin reactions in vivo [14].

The progress in applying molecular biology and genetic engineering in the last decade strongly contributed to the rapid development in the field of molecular allergology. The fastest method to clone an allergen is amplification of the coding sequence from reverse transcribed mRNA by PCR [15], [16], [17]. However, direct cloning of allergens by PCR requires sequence information, at least from the N-terminal amino acid sequence, which is not available in the normal case. Therefore, classical methods for the screening based on bacteriophage lambda [18], [19] have been adapted to screen cDNA expression libraries constructed with mRNA of different allergenic sources using serum IgE from sensitised individuals to detect allergen-expressing clones [20], [21], [22]. λ-based cloning strategies have been successful in generating information about the sequence of many allergens; however, efficient handling of large libraries is greatly facilitated if screening can be based on selective enrichment of clones expressing genes of interest [23], [24], [25]. The basic requirement for selective enrichment of clones consists in the physical linkage between genotype and phenotype [26], [27]. This requirement is not fulfilled by λ-based cDNA libraries where genetic information, integrated into the phage genome, and expressed gene products produced exploiting the machinery of the host cell during the lytic process, are not physically linked [28]. Therefore, these kinds of libraries require immobilisation to solid phase supports for screening, hampering selective enrichment of clones by specific gene-product/ligand interaction [18], [19], [24]. In contrast, filamentous phage engineered to display gene products on their coats contains the DNA encoding the displayed gene product as a part of their single-stranded genome [29], providing a physical link between genotype and phenotype (Fig. 1). This allows the survey of large libraries for the presence of specific clones using the discriminative power of affinity purification. In this contribution, we describe the application of phage display technology as an efficient method for rapid cloning of allergenic molecules.

Section snippets

Phage display technology

The basis of biological display technology relies on the physical link between the displayed entity and the gene coding for the displayed entity, thus connecting genotype and phenotype [30]. As a consequence thereof, it is possible to isolate members fulfilling specific criteria through an iterative “Darwinian” selection process from large and diverse repertoires cloned into filamentous phage [31]. Filamentous phages represent relatively simple biological systems, which are able to infect

Description of methods

The most common approach used to link phenotype and genotype has been to fuse the displayed molecule as an N-terminal extension of the viral pIII coat protein [34]. Although phage display has been used extensively for selection of peptides and antibodies, it has its limitations when applied to the expression of unknown sequences from cDNA libraries. As many of these sequences contain stop codons in their 3 untranslated regions, it is not feasible to directly fuse these sequences to the

Performance and limitation of phage display

The power of the phage display technology relies on the linkage of the functional display of molecular libraries with the ability of each member in the library to self-replicate and, at the same time, to encode the nucleotide sequence of the displayed polypeptide in its genome. The physical link between phenotype and genotype allows the fast isolation of desired clones through consecutive rounds of affinity enrichment [23], [42], [43]. The selection format is usually based on biopanning of the

Concluding remarks

Since the first description of phage display in 1985 [26], phage vectors have been used to display and select a variety of ligands, but the potential of phage as vectors for cDNA cloning was not a main focus of interest. Recently, phage vectors have been remodelled and developed for the expression of cDNA libraries fused to the gIII [23], [29], [40] or to the gVI genes [74] and used to clone genes of interest by affinity enrichment of phage displaying the desired gene products [50], [63], [64],

Acknowledgments

We are grateful to Professor Dr. K. Blaser for his continuous support. This work was supported by the Swiss National Science Foundation Grant No. 31-63381.00.

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