Enhancing multiple disulfide bonded protein folding in a cell‐free system

Medientyp: E-Artikel
Titel: Enhancing multiple disulfide bonded protein folding in a cell‐free system
Beteiligte: Yin, Gang; Swartz, James R.
Erschienen: Wiley, 2004
Erschienen in: Biotechnology and Bioengineering
Sprache: Englisch
DOI: 10.1002/bit.10827
ISSN: 0006-3592; 1097-0290
Schlagwörter: Applied Microbiology and Biotechnology ; Bioengineering ; Biotechnology
Entstehung:
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Beschreibung: <jats:title>Abstract</jats:title><jats:p>A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell‐free protein synthesis system. Due to the unstable and reducing environment in the initial <jats:italic>E. coli</jats:italic>‐based cell‐free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an <jats:italic>E. coli</jats:italic> periplasmic chaperone, and expressing PA at 30°C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 μg/mL of bioactive PA in a simple 3‐h batch reaction. © 2004 Wiley Periodicals, Inc.</jats:p>

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