Tapping allergen repertoires by advanced cloning technologies.

BACKGROUND: Complex allergenic sources such as moulds, foods and mites contain complex panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. METHODS: Phage surface display of cDNA libraries selectively enriched for allergen-expressing clones using IgE from allergic patients allows rapid isolation of large panels of allergens. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, high-throughput screening technology is required. RESULTS: The combination of selective enrichment of cDNA libraries based on biopanning against serum IgE from sensitized patients and automated robot technology for picking and high-density gridding of clones onto filter membranes, followed by hybridization, enables fast identification of all the different clones present in an enriched library. The consequent application of selective enrichment and robotic-based screening allows, within weeks, cloning and characterization of the whole allergenic repertoire of any organisms. CONCLUSIONS: Robotic-based high-throughput screening of clones selected for IgE-binding capacity from phage surface-displayed cDNA libraries of Aspergillus fumigatus, Cladosporium herbarum, Coprinus comatus, Malassezia furfur, peanut and human lung tissue allowed rapid characterization of 81, 28, 37, 27, 8 and 151 different sequences, respectively. All these cDNAs bear a high probability to encode allergens derived from the respective allergenic source.

High-throughput screening of surface displayed gene products.

With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.

Protein array technology. Potential use in medical diagnostics.

The human genome is sequenced, but only a minority of genes have been assigned a function. Whole-genome expression profiling is an important tool for functional genomic studies. Automated technology allows high-throughput gene activity monitoring by analysis of complex expression patterns, resulting in fingerprints of diseased versus normal or developmentally distinct tissues. Differential gene expression can be most efficiently monitored by DNA hybridization on arrays of oligonucleotides or cDNA clones. Starting from high-density filter membranes, cDNA microarrays have recently been devised in chip format. We have shown that the same cDNA libraries can be used for high-throughput protein expression and antibody screening on high-density filters and microarrays. These libraries connect recombinant proteins to clones identified by DNA hybridization or sequencing, hence creating a direct link between gene catalogs and functional catalogs. Microarrays can now be used to go from an individual clone to a specific gene and its protein product. Clone libraries become amenable to database integration including all steps from DNA sequencing to functional assays of gene products.

An ordered Arabidopsis thaliana mitochondrial cDNA library on high-density filters allows rapid systematic analysis of plant gene expression: a pilot study.

The availability of the complete sequence of a genome allows a systematic analysis of its expression. Gene-specific variations of transcription levels and phenomena such as transcript processing and RNA editing require large numbers of clones to be examined. For the completely sequenced mitochondrial genome of Arabidopsis thaliana we adapted robot technology to identify and characterize expressed genes. A cDNA library of about 50,000 clones was constructed, robot-ordered into 384-well microtitre plates and spotted onto high-density filter membranes. These filters permit the isolation of large numbers of specific cDNA clones in a single hybridization step. The cox1, cox2 and cox3 genes were used to evaluate the feasibility and efficiency of this approach. A cluster of RNA editing sites observed outside the cox3 coding region identifies a novel reading frame orf95 in higher plants with significant similarity to a subunit of respiratory chain complex II.