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首页> 外文期刊>Protein Expression and Purification >Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase
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Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

机译：甲苯4-单加氧酶的氧化还原酶成分的可溶性表达和纯化

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摘要

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 similar to 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained I mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed similar to 1 mol of the S = 1/2 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 KM- and apparent k(cat)-values of 0.15 mu M 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. (c) 2007 Elsevier Inc. All rights reserved.
机译：甲苯4-单加氧酶（T4MO）是细菌多组分单加氧酶的成员，该酶家族利用可溶性二铁羟化酶氧化多种烃类作为其代谢的第一步。羟化酶通过电子转移链从NAD（P）H获得还原当量，该电子转移链由包含N末端铁氧还蛋白域和C末端黄素和NAD结合域的氧化还原酶引发。 T4moF，即T4MO的NADH氧化还原酶，是从pUC衍生的表达载体pRS205在大肠杆菌BL21（DE3）中表达为可溶蛋白。该载体含有lac启动子而不是T7启动子。从可溶性细胞裂解物中进行的三步纯化得到的每克湿细胞糊剂的纯度接近90％，相当于1 mg T4moF。每摩尔T4moF纯化的蛋白质包含1摩尔的FAD和2摩尔的铁;定量EPR光谱显示，每摩尔T4moF与还原的[2Fe-2S]簇产生的1摩尔S = 1/2信号相似。以T4moF作为可变底物而所有其他蛋白质和底物保持恒定的对甲酚形成活性的稳态动力学分析得出的表观KM-值和表观k（cat）值分别为0.15μM和3.0 s（-1）。 。该表达系统和纯化允许以促进进一步生化和结构表征的产率回收可溶性氧化还原酶。 （c）2007 Elsevier Inc.保留所有权利。

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