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 ∼30 mg of crude PDO and ∼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 Escherichia 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 hours) 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.Among the various systems used for the expression of recombinant proteins, Escherichia coli has the advantage of being available in a wide array of mutant host strains, the ability to grow rapidly and to high density, of being better characterized genetically than other microorganisms, of having many compatible expression plasmids, and it gives good yields of target proteins. Considerable attention has been paid to the improvement of E. coli systems for expression of complex eukaryotic proteins (1,2). However, expression has been especially troublesome for proteins containing Fe-S clusters that are involved in a number of physiological processes including catalysis, electron transfer, biosynthesis, DNA repair and transcriptional regulation, and sensing for regulatory processes (3). In many cases supplementation of the growth media with Fe 2+ and S 2− was not sufficient for good expression (3). The formation of Fe-S clusters was found to depend on the so-called isc (iron-sulfur cluster) genes (4,5), all nine individual components of which are important for the expression of some active ). In a number of cases it was found that coexpression of the isc cluster was essential for achieving high yields of active recombinant Fe-S proteins (11-13). Cluster inactivation in E. coli resulted in a marked decrease in the production of native and recombinant iron-sulfur * To whom correspondence should be addressed. Email: dballou@umich.edu Publisher's Dis...

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

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Biosynthesis of 8-O-methylated benzoxazinoid defense compounds in maize

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