In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell-based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost-optimized, scalably-manufactured Escherichia coli extract and minimal supplement reagents to perform cell-free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti-GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1-7)M(2-9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.
Keywords: cell-free protein synthesis; linear template; rolling circle amplification.
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