Abstract
Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.
Highlights
Proteins have increasing medical, industrial and research importance, owing to their high versatility, bio-specificity and potency [1]
We present here a quick, inexpensive and simple procedure for lysate preparation and in vitro protein production
The system exhibited improved activity in temperatures higher than 37°C, depending on the bacteria strain, which can be an advantage for different applications
Summary
Industrial and research importance, owing to their high versatility, bio-specificity and potency [1]. There is a growing need for efficient and economical methods for producing proteins [2,3,4]. Expressing proteins in yeast, bacteria or mammalian cells is usually the first choice; for some proteins this is impossible and other expression systems are needed [5]. Protein synthesis in a cell free system can allow the production of toxic proteins, the incorporation of artificial amino acids, and high-throughput screening of proteins [6]. CFPS systems can be prepared from either a cell extract, or from a combination of purified recombinant proteins [7,8,9]. CFPS systems based on E. coli extracts, termed ‘S30
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