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.

Establishing a high yielding streptomyces-based cell-free protein synthesis system.

Cell-free protein synthesis (CFPS) systems enable the production of protein without the use of living, intact cells. An emerging area of interest is to use CFPS systems to characterize individual elements for genetic programs [e.g. promoters, ribosome binding sites (RBS)]. To enable this research area, robust CFPS systems must be developed from new chassis organisms. One such chassis is the Gram-negative Pseudomonas bacteria, which have been studied extensively for their diverse metabolism with promises in the field of bioremediation and biosynthesis. Here, we report the development and optimization of a high-yielding (198 ± 5.9 µg/ml) batch CFPS system from Pseudomonas putida ATCC 12633. Importantly, both circular and linear DNA templates can be applied directly to the CFPS reaction to program protein synthesis. Therefore, it is possible to prepare hundreds or even thousands of DNA templates without time-consuming cloning work. This opens the possibility to rapidly assess and validate genetic part performance in vitro before performing experiments in cells. To validate the P. putida CFPS system as a platform for prototyping genetic parts, we designed and constructed a library consisting of 15 different RBSs upstream of the reporter protein sfGFP, which covered an order of magnitude range in expression. Looking forward, our P. putida CFPS platform will not only expand the protein synthesis toolkit for synthetic biology but also serve as a platform in expediting the screening and prototyping of gene regulatory elements.

Development of a Pseudomonas putida cell-free protein synthesis platform for rapid screening of gene regulatory elements

TRIM21 and the Function of Antibodies inside Cells.

Cell-free protein synthesis: Recent advances in bacterial extract sources and expanded applications

Stratified medicines are defined as medicines which target diseases where the patients have been preselected for treatment based on their response to a diagnostic test. The pipeline of these medicines cover a wide range of different treatment types including cell and gene therapies, vaccines based on peptides or proteins; and protein based therapies. These increasingly diverse and by definition smaller market size products require improved agility and productivity in process design if manufacture and supply of affordable medicines is to be achieved. In this paper we review the current state of cell free synthesis (CFS), the new technologies and strategies being developed and its application to the production of stratified medicines; focusing on the production of protein based therapeutic products.

Cell free protein synthesis: a viable option for stratified medicines manufacturing?

Antibodies that target intracellular proteins hold great promise in the development of novel therapeutic interventions for various diseases. In particular, antibodies that can cross cellular membranes have potential applications in controlling disease-related intracellular protein-protein interactions. Given the large number of cytosolic proteins and complicated interactions that are potentially involved in disease development, discovery of antibodies targeting intracellular proteins requires iterative cycles of expression and assessment of candidate antibodies. Because current cell-based expression methods do not provide sufficient throughput for production and assay of cytosol-penetrating antibodies, we integrated a cell-free protein synthesis system designed to provide optimal conditions for production of functional antibodies with a cytosol-penetration assay. The proposed approach of consolidating cell-free synthesis and cell-based assay will substantially expand the capability of discovering and engineering antibodies that can cross the cell membrane and effectively control protein-mediated cellular functions. Biotechnol. Bioeng. 2016;113: 2107-2112. © 2016 Wiley Periodicals, Inc.

Cell-free production and streamlined assay of cytosol-penetrating antibodies.

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Cell-free production and streamlined assay of cytosol-penetrating antibodies.