A highly efficient and economical cell-free protein synthesis system using the S12 extract of Escherichia coli

Abstract

We have developed an economical and simple cell-free protein synthesis system that produces milligram quantities of proteins in a milliliter batch reaction. In this system, the S12 extract, which was prepared from glucose-adapted cells, was employed and glucose alone was successfully used for the efficient and stable regeneration of ATP. The ATP level in the reaction mixture remained stable over a remarkably extended reaction period, which enabled prolonged protein synthesis, and the issues associated with proton accumulation and amino acid depletion were simultaneously addressed. Under the reaction conditions established in this study, protein synthesis continued for 6 h and the amount of the accumulated protein reached 1.8 mg/mL. © KSBB hÉóïçêÇëW=ÅÉääJÑêÉÉ=éêçíÉáå=ëóåíÜÉëáëI=ÖäìÅçëÉI=ÅÜäçê~ãéÜÉåáÅçä=~ÅÉíóäíê~åëÑÉê~ëÉI=k^aI=`ç^I=éeI=ÅóëíÉáåÉ= = = = =

Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage

“…Growths were induced with 1 mM IPTG (GoldBio, St Louis, MO) at an OD 600 of 0.40.7 and grown until mid- to late-log phase (an OD 600 of 2.0 in this work). Cells were harvested, washed, homogenized, and prepared using the streamlined method described previously (3, 27). …”

Advances in Protein Production Technologies

“…In contrast to in vivo systems, cell -free methods are amenable to direct condition monitoring, and provide greater control for substrate and product transportation. Cell -free extracts are most commonly prepared from E. coli cells [187,188] and wheat germs [189,190] . Rabbit reticulocyte lysates are used [191] to a lesser extent due to their inability to produce preparative amounts of proteins other than globins.…”

“…To prepare E. coli lysates, the cells are cultivated until mid -log phase, cooled, and harvested by centrifugation [187,188,193,194] . Collected cells are homogenized and cell debris removed by centrifugation at 30 000 g .…”

Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source

“…The productivity and economics of cell-free protein synthesis have also been improved remarkably during the past decade, and production of milligram amounts of protein is now possible [2][3][4][5][6][7][8]. These advances have primarily been possible due to implementation of more efficient strategies for ATP regeneration during protein synthesis reactions.…”

“…The rate at which the pH decreased was sufficient to inhibit protein synthesis [7,8], compromising the benefits of using glucose.…”