期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 52, 期 2, 页码 273-279出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2006.09.004
关键词
iron-sulfur protein; Rieske center; phthalate dioxygenase; high yield expression
类别
资金
- NCRR NIH HHS [P41 RR004328-100022] Funding Source: Medline
- NIGMS NIH HHS [R37 GM020877, R01 GM020877, F32 GM020877, R01 GM020877-31A1, GM20877] Funding Source: Medline
Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II)-mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type fierredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at similar to 30 mg of crude PDO and similar to 1 mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to E coli cell line, growth parameters, time of induction, media composition, and iron-sulfur additives resulted in yields of about 1 g/L for PDO and about 0.2 g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20 h) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5-9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was < 8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein. (c) 2006 Elsevier Inc. All rights reserved.
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