Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli |
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Authors: | Kim Dong-Myung Swartz James R |
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Institution: | Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA. |
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Abstract: | 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. |
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Keywords: | cell‐free protein synthesis urokinase disulfide bond formation protein folding sulfhydrylredox potential |
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