Cell-free protein synthesis: methodology and applications in structural and synthetic biology

TOPICAL COLLECTIONS

Guest Editors

Dr Nadanai Laohakunakorn

University of Edinburgh, Institute of Quantitative Biology, Biochemistry, and Biotechnology, School of Biological Sciences

Dr. Nadanai Laohakunakorn, originally from Bangkok, obtained his BA/MSci in Natural Sciences (Physics) from Trinity College, Cambridge in 2010. He remained in Cambridge to carry out his PhD under the supervision of Prof. Ulrich Keyser, where he studied nanopores and single-molecule biophysics. After defending his thesis in 2015, he moved to Lausanne where he worked with Prof. Sebastian Maerkl at EPFL on cell-free synthetic biology. His work there focused on combining microfluidic devices with cell-free gene expression systems for high-throughput and rapid prototyping of genetic parts and circuits. In 2019 he received a Chancellor's Fellowship in Biotechnology at the University of Edinburgh, where he has established a lab for quantitative engineering of cell-free systems. His current research interests include coupling synthetic metabolism with cell-free protein synthesis, regenerative cell-free systems, and metabolomic analysis and computational approaches for the optimization of cell-free reactions.

Ray Owens

University of Oxford

Ray Owens has extensive experience of the production of recombinant proteins including engineered antibodies both in industry and academia. He obtained his PhD in biochemistry at the University of Cambridge, UK, and has a long- standing interest in protein structure and function. He is currently a Professor of Molecular biology at the University of Oxford and Head of the Protein Production Facility in the Research Complex at Harwell (RCaH). RCaH is a multi-disciplinary Research Institute, based at the Rutherford Appleton Laboratory, near Oxford.

Collection Overview

Coupled in vitro transcription – translation systems (IVTT) enable the cell-free synthesis of a wide variety of proteins, including integral membrane proteins, and is particularly useful in the production of proteins that may be toxic to cells.

Cell-free systems can be generated using lysates from different source organisms, including bacterial, insect, plant and mammalian cells, or by using reconstituted systems composed of purified recombinant elements. These are incubated with plasmids or linear PCR products consisting of a promoter, typically T7, coupled to the gene of interest. As open systems, IVTT reactions can be supplemented with ligands or additional cofactors to improve yields and introduce novel functionality, for example incorporation of non-natural amino acids.

In structural biology, IVTT has traditionally been used to label proteins for NMR studies and with advances in cryo-electron microscopy may also have a role in the production of large protein complexes for single particle analysis.

IVTT has become a powerful tool in synthetic biology for rapid prototyping and testing of genetic circuits and metabolic pathways, as well as the development of applications such as biosensors for environmental monitoring, and low-cost distributed biomanufacturing platforms. Active areas of research related to synthetic cells involve combining cell-free synthesis with compartmentalization, and studying regeneration of system components as well as communication between compartments.

This collection aims to bring together and disseminate methods relevant to a variety of applications of IVTT in structural and synthetic biology, including production of different classes of proteins, as well as the design, construction, analysis, and optimization of cell-free genetic and metabolic circuits and pathways.