Alexei M. Voloshin

An integrated cell‐free metabolic platform for protein production and synthetic biology

Cell‐free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell‐free systems, however, have been limited by their inability to co‐activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell‐free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co‐activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell‐free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.

Expression of Active Murine Granulocyte‐Macrophage Colony‐Stimulating Factor in an Escherichia coli Cell‐Free System

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM‐CSF in an E. coli cell‐free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM‐CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM‐CSF was found in its oxidized form. However, the GM‐CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM‐CSF was produced in the cell‐free reaction. Finally, successful folding of the cell‐free synthesized GM‐CSF‐his6 was confirmed by its cell‐proliferation activity after purification with a Ni2+ chelating column.