FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems

Abstract

Abstract Backgroud Cell-free protein synthesis systems (CFPS) have a wide range of applications ranging from educational to high-throughput screening. The detection of proteins in CFPS is accomplished through various methods, each with its own limitation: the use of radioactive labeling has become impractical for many laboratories due to the disposal costs, the incorporation of fluorescent tags often demands both costly and time-intensive procedures and the synthesis of large target-reporter fusions may be challenging owing to the limitation of the substrates. Results The Green Fluorescent Protein (GFP) can reassemble from two fragments (split-GFP): a large fragment called GFP 1-10 and a small fragment called GFP11. Here, we developed the FAST (Fluorescent Assembly of Split-GFP for Translation Tests) method to monitor protein synthesis in CFPS. FAST relies on the fusion of the small tag GFP11 to virtually any gene to be expressed in CFPS. The in vitro synthesized protein:GFP11 can be rapidly detected in solution upon interaction with an enhanced GFP1-10 fused to the Maltose Binding Protein (MBP:GFP1-10) using a fluorescent reader. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. To demonstrate the feasibility and reproducibility of the system, four E. coli genes of increasing length were fused to the GFP11 fragment and tested using FAST. Protein synthesis was carried out using both an in-house E. coli crude extract and a commercial E. coli reconstituted system for coupled transcription/translation. Our results demonstrate that FAST develops a fluorescent signal that is proportional to the amount of the synthetized protein:GFP11 fusions, with an estimated sensitivity of 8±2 pmoles of polypeptide. Fluorescence develops rapidly and plateaus after 4 hours. In addition, FAST allows to monitor antibiotic-dependent inhibition of translation in a concentration-dependent way. Conclusions FAST is a novel method for rapidly and easily tracking cell-free protein synthesis avoiding radiolabeling or electrophoretic separation. FAST is particularly suitable for screening panels of genes and factors/bioactive metabolites that influence translation, as well as in research areas where the products of CFPS are required for downstream analysis or testing, such as in the synthetic biology or protein design field.