In Situ Biosynthesis of Peptide Arrays

Polypeptide and protein arrays enable high-throughput screening capabilities for studying molecular interactions and profiling of biomarkers, and provide a powerful functional screening tool for peptidomics. To overcome the limitations of conventional arraying methods, we have exploited cell-free systems for generating arrays of polypeptides by direct on-chip biosynthesis from DNA templates. Here we describe two protocols: (i) Protein In Situ Array (PISA), which allows the generation of polypeptide arrays in a single reaction by spotting cell-free lysate together with PCR DNA on a glass surface pre-coated with a capturing reagent, and (ii) DNA Array to Protein Array (DAPA), which is capable of producing multiple copies of a polypeptide array from a single DNA array template. The main advantage of these methods is in using an in vitro coupled transcription and translation system which circumvents the need to synthesise and purify individual polypeptides. Our methods allow making polypeptide arrays using amplified linear DNA fragments.