Imperial College London; Hammersmith Campus 1 article published in JoVE Bioengineering A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications Ming Toh1,2,3,4, Kameshwari Chengan5, Tanith Hanson5, Paul S. Freemont1,2,3,4,6,7, Simon J. Moore5 1Centre for Synthetic Biology and Innovation, South Kensington Campus, 2Department of Medicine, South Kensington Campus, 3Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, 4Sir Alexander Fleming Building, South Kensington Campus, 5School of Biosciences, Division of Natural Sciences, University of Kent, 6UK Dementia Research Institute Care Research and Technology Centre, Imperial College London; Hammersmith Campus, 7UK Innovation and Knowledge Centre for Synthetic Biology (SynbiCITE) and the London Biofoundry, Imperial College Translation & Innovation Hub This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.