RApid Parallel Protein EvaluatoR (RAPPER), from gene to enzyme function in one day

Abstract

Cell-free transcription–translation systems offer an effective and versatile platform to explore the impact of genetic variations on protein function. We have developed a protocol for preparing linear, mutagenic DNA templates for direct use in the PURE system, enabling the fast and semi-quantitative evaluation of amino acid variations on catalytic activity and stereo-selectivity in native and engineered variants of Old Yellow Enzyme.