Optimized Expression and Purification of Toluene 4-Monooxygenase Hydroxylase

Studts, Joey; Mitchell, Kevin H.; Pikus, Jeremie D.; McClay, Kevin; Steffan, Robert J.; Fox, Brian G.

doi:10.1006/prep.2000.1281

Cited by 31 publications

(28 citation statements)

References 32 publications

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“…In contrast, the use of pT4moABEF gave reproducible soluble expression, albeit at a lower level. This is a pUC18-derived expression vector that has a high copy number [5], which leads to significant basal expression from the lac promoter [5]. Thus addition of IPTG to cultures containing pT4moABEF gave only ∼2-fold in the expression level, with much of the expression arising during the 24 h period of culture scale-up; this can be contrasted to the 20-to 100-fold increase observed with the pET vectors in an ∼4 h period.…”

Section: Expression Contextmentioning

confidence: 97%

“…Previous studies on expression from pT4moABEF demonstrated that it is not a suitable vector for overexpression of the T4moH component [5]. Thus the yield of purified T4moH from pT4moABEF (1.9 mg·g -1 of cell paste) was typically only 30% of that from pKM11 (6.2 mg·g -1 of cell paste), another pUC18-based vector.…”

Section: Expression Contextmentioning

confidence: 98%

“…Previous efforts on the expression of T4moC, T4moD, and T4moH (TmoA, TmoB, and TmoE polypeptides) have yielded greater than 50 mg of purified, soluble protein per liter culture medium [5]. However, this level of soluble expression has not been achieved with T4moF, which hindered the purification and subsequent characterizations and functional studies.…”

Section: Expression Of T4mofmentioning

confidence: 99%

“…Moreover, a natural rho-independent terminator immediately before the start codon of the tmoF gene attenuated the expression [3]. The expression medium was supplemented with Casamino acids and riboflavin; other experiments have shown this medium has sufficient iron to support cofactor incorporation at this cell density [5]. The expression was carried out at 25 °C, and the expression was terminated at low cell density.…”

Section: Expression Contextmentioning

confidence: 99%

“…The hydroxylase (T4moH), effector protein (T4moD) and Rieske-type ferredoxin (T4moC) of the T4MO system have been purified and extensively characterized [2,[5][6][7][8][9][10][11][12][13]. In contrast, study of T4moF has been limited by difficulties in obtaining soluble expression, with subsequent limits on the ability to obtain purified protein.…”

Section: Introductionmentioning

confidence: 99%

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Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Bailey

Elsen

Pierce

et al. 2008

Protein Expression and Purification

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Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin-and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded ∼1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitiative EPR spectroscopy showed ∼1 mol of the S = ½ signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K M -and apparent k cat -values of 0.15 μM and 3.0 s -1 , respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.

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Section: Expression Contextmentioning

confidence: 97%

Section: Expression Contextmentioning

confidence: 98%

Section: Expression Of T4mofmentioning

confidence: 99%

Section: Expression Contextmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Bailey

Elsen

Pierce

et al. 2008

Protein Expression and Purification

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Toluene 4‐Monooxygenase and its Complex with Effector ProteinT4moD

Bailey

Fox

2004

Handbook of Metalloproteins

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Toluene 4‐monooxygenase (T4MO) is a multiprotein diiron enzyme complex that catalyzes the regiospecific oxidation of toluene to p ‐cresol. Catalytic function requires the presence of a small protein, called the effector protein . Effector protein exerts substantial control on the diiron hydroxylase catalytic cycle through protein–protein interactions. High‐resolution crystal structures of the stoichometric hydroxylase and effector protein complex described here reveal how protein–protein interactions and reduction of the diiron center produce an active site configuration poised for reaction with O 2 . Further information from crystal structures of mutated isoforms of the hydroxylase and a peroxo adduct is combined with catalytic results to give a fuller picture of the geometry of the enzyme–substrate complex used for the high fidelity oxidation of hydrocarbon substrates.

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Autoinduction of Protein Expression

Fox

Blommel

2009

CP Protein Science

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This unit contains protocols for the use of lactose-derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X-ray structural studies, and multi-well plate growth for screening and optimization are also included.

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Optimized Expression and Purification of Toluene 4-Monooxygenase Hydroxylase

Cited by 31 publications

References 32 publications

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Toluene 4‐Monooxygenase and its Complex with Effector ProteinT4moD

Autoinduction of Protein Expression

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