Cell-free synthesis of proteins that require disulfide bonds using glucose as an energy source.
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.
Knapp
Kurtis G
KG
Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
Goerke
Aaron R
AR
Swartz
James R
JR
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002474:Cell-Free System; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000201:enzymology; D004220:Disulfides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D005978:Glutathione / Q000032:analysis; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D051379:Mice; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D017510:Protein Folding; D013879:Thioredoxins / Q000032:analysis; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis / Q000096:biosynthesis*
D004220:Disulfides; D004791:Enzyme Inhibitors; D013879:Thioredoxins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D010088:Oxidoreductases; D014568:Urokinase-Type Plasminogen Activator; D005978:Glutathione; D005947:Glucose; D007460:Iodoacetamide