High throughput recombinant protein production of fungal secreted proteins

Secreted proteins are important for both symbiotic and pathogenic interactions between fungi and their hosts. Our research group uses screens and genomic mining to discover novel proteins involved in these processes. To efficiently study the large number of candidate proteins, we are establishing a high-throughput protein production system with a special focus on fungal secreted proteins. We use a ligation independent cloning to clone target genes into expression vectors for E. coli and P. pastoris and a small scale test expression to identify constructs producing soluble protein. Expressed soluble proteins are then produced in larger quantities, purified and assayed for new enzymatic activities. We used transposon-assisted signal sequence trapping (TAST) to identify putative secreted proteins expressed during the interactions between the basidiomycete Paxillus involutus and birch (symbiotic interaction), between fungi of the order Entomophthorales and aphids (pathogenic interaction), and in the mycoparasitic interaction between the oomycetes Pythium oligandrum and P. ultimum. In general, the high-throughput protein production system can lead to a better understanding of fungal/host interactions and can also identify potential industrially useful enzymes.