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

Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS. By harnessing the reassembly capabilities of two GFP fragments-specifically, the GFP1-10 and GFP11 fragments-we have crafted a method that simplifies the detection of in vitro synthesized proteins called FAST (Fluorescent Assembly of Split-GFP for Translation Tests). 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). This interaction produces a fluorescent signal detectable with standard fluorescence readers, thereby indicating successful protein synthesis. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. The method's versatility was demonstrated by fusing GFP11 to four distinct E. coli genes and analyzing the resulting protein synthesis in both a homemade and a commercial E. coli CFPS system. Our experiments confirmed that the FAST method offers a direct correlation between the fluorescent signal and the amount of synthesized protein:GFP11 fusion, achieving a sensitivity threshold of 8 ± 2 pmol of polypeptide, with fluorescence plateauing after 4 h. Additionally, FAST enables the investigation of translation inhibition by antibiotics in a dose-dependent manner. In conclusion, FAST is a new method that permits the rapid, efficient, and non-hazardous detection of protein synthesized within CFPS systems and, at the same time, the purification of the target protein.

Recent research into healthcare professions regulation: a rapid evidence assessment.

BACKGROUND AND AIMS: Over the last decade, regulators have taken significant steps towards tackling perceptions that regulatory systems are burdensome. There has been much international research activity in the regulation of health and care professionals. This article reports a review of studies on health professions regulation between January 2011 and March 2020. Its chief object was to provide robust and up-to-date evidence to assist regulators in policy development and implementation. The main objectives of this study were to: 1. Identify and retrieve research in the field of health and care professions regulation in English since 2011; 2. Evaluate the published research, exploring its utility to regulators and practitioners, and drawing out any key messages; 3. Draw conclusions concerning the scope and limitations of the research literature and identify areas for further research. METHODS: We undertook a rapid evidence assessment (REA) of the international literature on health and care professions regulation, including reviewing ten UK regulators' websites to identify issues of concern and strategic priorities. We retrieved 3833 references, using a four-stage screening process to select the 81 most relevant. RESULTS: Results are reported within six key themes: harm prevention and patient safety; fitness to practise; quality assurance of education and training; registration including maintenance of registers; guidelines and standards and relations with regulatory bodies. CONCLUSIONS: Regulation of professionals in health and care is comparatively undeveloped as a field of academic study. Consequently, the published evidence is diffuse and small-scale. Most work presents relatively weak data of low relevance to regulators, mainly reporting or describing the current position. Few studies are able to show the impact of regulation or demonstrate a causal link between regulation and its effects. To inform their research and policy agendas health and social care regulators need to commission, interpret and apply the scholarly literature more effectively; academics need to engage with regulators to ensure that their research provides high-quality evidence with practical relevance to the regulators' agendas. Further study is needed to explore how effective academic collaborations between regulators and researchers may be created and sustained.