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

Abstract

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 cellfree 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.

Continued Protein Synthesis at Low [ATP] and [GTP] Enables Cell Adaptation during Energy Limitation

“…Previously, we showed that approximately 100% of the CAT produced during the initial protein synthesis period (the first hour) is active (21,22). Thus, in the experiments performed here, the TCA-precipitable counts were a good indication of the active protein concentration.…”

“…The fraction of incorporated leucine was obtained by dividing the cpm of the precipitated sample by the cpm of the nonprecipitated sample. To illustrate, the following is a sample calculation if the TCA-precipitated count was 950 cpm, the nonprecipitated count was 9,996 cpm, and the background count from an experiment without plasmid DNA was 73 cpm (Table 1): {[(950 cpm Ϫ 73 cpm)/9,996 cpm] ϫ 2,000 M ϫ 25,000 g/mol}/(13 ϫ 1,000 ml/liter) ϭ 337 g CAT/ml.Previously, we showed that approximately 100% of the CAT produced during the initial protein synthesis period (the first hour) is active (21,22). Thus, in the experiments performed here, the TCA-precipitable counts were a good indication of the active protein concentration.…”

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

“…Here, we extend a scalable in vitro biochemical protein synthesis system, termed open cell-free synthesis (OCFS), 18,19 to the production of correctly folded antibody fragments and an aglycosylated version of anti-HER2 IgG1 trastuzumab. In vitro protein synthesis systems whereby a cell extract from E. coli cells harvested in exponential phase serves as a source of ribosomes and other cellular factors for translation 20 offer an alternative expression system that avoids many of the problems of conventional cell-based expression technologies.…”

A synthetic biochemistry molecular purge valve module that maintains redox balance

“…Improvements in protein expression and access to stable enzymes have made more complex systems possible, however 1,[9][10][11][12] . In vitro biotransformation systems have been reported in recent years involving systems of over 30 enzymes 13,14 .…”