Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli.
In this report, we demonstrate that a complex mammalian protein containing multiple disulfide bonds is successfully expressed in an E.coli-based cell-free protein synthesis system. Initially, disulfide-reducing activities in the cell extract prevented the formation of disulfide bonds. However, a simple pretreatment of the cell extract with iodoacetamide abolished the reducing activity. This extract was still active for protein synthesis even under oxidizing conditions. The use of a glutathione redox buffer coupled with the DsbC disulfide isomerase and pH optimization produced 40 microg/mL of active urokinase protease in a simple batch reaction. This result not only demonstrates efficient production of complex proteins, it also emphasizes the control and flexibility offered by the cell-free approach.
Kim
Dong-Myung
DM
Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
Swartz
James R
JR
D002457:Cell Extracts / Q000737:chemistry / Q000302:isolation & purification; D002474:Cell-Free System; D004220:Disulfides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D005240:Feasibility Studies; D007460:Iodoacetamide / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D002457:Cell Extracts; D004220:Disulfides; D029968:Escherichia coli Proteins; D014568:Urokinase-Type Plasminogen Activator; D007460:Iodoacetamide