Cell‐free synthesis of proteins that require disulfide bonds using glucose as an energy source

Abstract

The primary objective of this work was to create a cell-free protein synthesis extract that produces proteins requiring disulfide bonds while using glucose as an energy source. We attempted to avoid using iodoacetamide (IAM) to stabilize the required oxidizing thiol redox potential, since previous IAM pretreatments prevented glucose utilization apparently by inactivating glyceraldehyde 3-phosphate dehydrogenase (G-3PDH). Instead, the glutathione reductase (Gor)-mediated disulfide reductase system was disabled by deleting the gor gene from the KC6 cell-extract source strain. The thioredoxin reductase (TrxB)-mediated system was disabled by first adding a purification tag to the trxB gene in the chromosome to create strain KGK10 and then by affinity removal of the tagged TrxB. This was expected to result in a cell extract devoid of all disulfide reductase activity, but this was not the case. Although the concentration of IAM required to stabilize oxidized glutathione in the KGK10 extract could be reduced 20-fold, IAM pretreatment was still required to avoid disulfide reduction. Nonetheless, active urokinase and murine granulocyte macrophage-colony stimulating factor (mGM-CSF) were produced in reactions with KGK10 extract either with affinity removal of TrxB or with 50 microM IAM pretreatment. With the less intensive IAM pretreatment, glucose could be used as an energy source in a production system that promotes oxidative protein folding. This new protocol offers an economically feasible cell-free system for the production of secreted mammalian proteins as human therapeutics or vaccines.

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

“…The quality of E. coli extracts also has been improved in several ways, including optimization of extract preparation methods and engineering of an E. coli KC6 strain that maintains stable pools of amino acids during the protein synthesis [9,61]. The KC6 strain was then further engineered in favor of synthesis of disulWde bonded proteins by creating an optimal thiol redox potential [52]. At this point, the system has demonstrated a variety of applications, including production of protein therapeutics such as murine granulocyte macrophage colony stimulating factor (GM-CSF), scFvs and IGF-I.…”

Efficient disulfide bond formation in virus-like particles

“…Normally the CFPS environment actively reduces disulfide bonds due to the activity of soluble reductases from the E. coli cytosol (predominantly glutathione reductase, Gor, and thioredoxin reductase, TrxB (Knapp, 2006)). As reported previously when the Hepatitis B core antigen VLP (HBc VLP) was produced using CFPS, it lacked the C61 intermolecular disulfide linkage that bridges the dimer (Scheme 2C) (Bundy et al, 2008).…”

Hazards, Risks, and Low Hazard Development Paths of Synthetic Biology

“…Furthermore, convenient strategies for trophic and semantic containment still 28 Interfering background reactions as a cause for perturbed functions or diminished product recovery rates can occur in cellular extracts as well. However, extracts can be improved by mutation and selection of the required strains (Knapp et al 2007). 29 Hockenberry and Jewett (2012, 257) also mention the benefits for standardized elements in synthetic biology: "While the search for biological 'parts' has proven fruitful for in vivo synthetic biologists, many of these parts are still highly context dependent.…”