Journal Article10.1002/BIT.26253
Establishing a high yielding streptomyces-based cell-free protein synthesis system.
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TL;DR: It is shown that the optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli‐based CFPS system for increasing percent soluble protein expression for four StrePTomyces‐originated high GC‐content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin.
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Abstract: Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc.
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Cell-free gene expression: an expanded repertoire of applications.
TL;DR: Advances provide exciting opportunities to profoundly transform synthetic biology by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up, and next-generation educational kits.
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A User's Guide to Cell-Free Protein Synthesis.
Nicole E. Gregorio,Max Z. Levine,Javin P. Oza +2 more
- 12 Mar 2019
TL;DR: This review will help to contextualize the landscape of work that has been done using CFPS and showcase the diversity of applications that it enables and clarify the similarities and differences amongst cell-free platforms.
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Cell-free gene expression
David Garenne,Matthew C. Haines,Eugenia F. Romantseva,Paul S. Freemont,Elizabeth A. Strychalski,Vincent Noireaux +5 more
- 15 Jul 2021
TL;DR: This Primer provides new practitioners with a comprehensive, detailed and actionable guide to best practices in CFE, to inform research in the laboratory at the state of the art.
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Kara Calhoun,James R. Swartz +1 more
TL;DR: This work created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization, and confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase in the cell extract.
Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines
TL;DR: These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin.
Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system
TL;DR: The development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase that can be prepared quickly within 4–5 h and represents a remarkable improvement over current eukaryotic cell- free systems.
Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-Free System
TL;DR: Methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation, are characterized and used to use linear DNA PCR products in TX-TL.