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

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Cell-free transcription-translation systems provide a flexible platform for studying protein function.
• Genetic variations can significantly alter protein properties, necessitating efficient evaluation methods.

Purpose of the Study:

• To develop a protocol for preparing linear DNA templates for cell-free systems.
• To enable fast and semi-quantitative assessment of amino acid variations in proteins.
• To evaluate catalytic activity and stereo-selectivity of Old Yellow Enzyme variants.

Main Methods:

• Development of a protocol for linear, mutagenic DNA template preparation.
• Utilizing the PURE system (Protein synthesis Using Recombinant Elements) for cell-free expression.
• Semi-quantitative evaluation of enzyme variants' catalytic activity and stereo-selectivity.

Main Results:

• A protocol for preparing linear DNA templates compatible with the PURE system was established.
• The method allows for rapid assessment of amino acid variations.
• The impact of variations on Old Yellow Enzyme's catalytic activity and stereo-selectivity was evaluated.

Conclusions:

• The developed protocol offers an efficient approach for analyzing genetic variations' effects on protein function.
• This method facilitates the study of both native and engineered enzyme variants.
• Cell-free systems coupled with optimized DNA templates are powerful tools for protein engineering and functional studies.