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Efficient production of a bioactive, multiple disulfide‐bonded protein using modified extracts of Escherichia coli
Author(s) -
Kim DongMyung,
Swartz James R.
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10865
Subject(s) - iodoacetamide , protein disulfide isomerase , cell free protein synthesis , chemistry , biochemistry , oxidizing agent , disulfide bond , glutathione , reducing agent , combinatorial chemistry , protease , tcep , cysteine , enzyme , protein biosynthesis , organic chemistry , phosphine , catalysis
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 μg/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. © 2003 Wiley Periodicals, Inc.