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High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli.

Abstract：

: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 ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at approximately 30mg of crude PDO and approximately 1mg 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 1g/L for PDO and about 0.2g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20h) 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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Jaganaman S,Pinto A,Tarasev M,Ballou DP

doi

10.1016/j.pep.2006.09.004

subject

Has Abstract

pub_date

2007-04-01 00:00:00

pages

273-9

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(06)00289-0

journal_volume

52

pub_type

杂志文章

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