Urarte Estibaliz, Auzmendi Iñigo, Rol Selene, Ariz Idoia, Aparicio-Tejo Pedro, Arredondo-Peter Raúl, Moran Jose F
Instituto de Agrobiotecnologia, Universidad Pública de Navarra-CSIC-Gobierno de Navarra, Pamplona, Navarre, Spain.
Methods Enzymol. 2008;436:411-23. doi: 10.1016/S0076-6879(08)36023-6.
Ferric leghemoglobin reductase (FLbR) is able to reduce ferric leghemoglobin (Lb3+) to ferrous (Lb2+) form. This reaction makes Lb functional in performing its role since only reduced hemoglobins bind O2. FLbR contains FAD as prosthetic group to perform its activity. FLbR-1 and FLbR-2 were isolated from soybean root nodules and it has been postulated that they reduce Lb3+. The existence of Lb2+ is essential for the nitrogen fixation process that occurs in legume nodules; thus, the isolation of FLbR for the study of this enzyme in the nodule physiology is of interest. However, previous methods for the production of recombinant FLbR are inefficient as yields are too low. We describe the production of a recombinant FLbR-2 from Escherichia coli BL21(DE3) by using an overexpression method based on the self-induction of the recombinant E. coli. This expression system is four times more efficient than the previous overexpression method. The quality of recombinant FLbR-2 (based on spectroscopy, SDS-PAGE, IEF, and native PAGE) is comparable to that of the previous expression system. Also, FLbR-2 is purified near to homogeneity in only few steps (in a time scale, the full process takes 3 days). The purification method involves affinity chromatography using a Ni-nitrilotriacetic acid column. Resulting rFLbR-2 showed an intense yellow color, and spectral characterization of rFLbR-2 indicated that rFLbR-2 contains flavin. Pure rFLbR-2 was incubated with soybean Lba and NADH, and time drive rates showed that rFLbR-2 efficiently reduces Lb3+.
铁血红蛋白还原酶（FLbR）能够将高铁血红蛋白（Lb3+）还原为亚铁血红蛋白（Lb2+）形式。由于只有还原型血红蛋白才能结合氧气，因此该反应使血红蛋白能够发挥其功能。FLbR含有黄素腺嘌呤二核苷酸（FAD）作为辅基来发挥其活性。FLbR - 1和FLbR - 2是从大豆根瘤中分离出来的，据推测它们能还原Lb3+。Lb2+的存在对于豆科植物根瘤中发生的固氮过程至关重要；因此，分离FLbR以研究该酶在根瘤生理学中的作用具有重要意义。然而，以前生产重组FLbR的方法效率低下，因为产量太低。我们描述了通过基于重组大肠杆菌自诱导的过表达方法，从大肠杆菌BL21(DE3)中生产重组FLbR - 2。该表达系统的效率比以前的过表达方法高四倍。重组FLbR - 2的质量（基于光谱学、十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）、等电聚焦（IEF）和非变性聚丙烯酰胺凝胶电泳（native PAGE））与以前的表达系统相当。此外，FLbR - 2只需几步就能纯化至接近均一性（在时间尺度上，整个过程需要3天）。纯化方法包括使用镍 - 次氮基三乙酸柱进行亲和色谱。所得的重组FLbR - 2呈现出强烈的黄色，重组FLbR - 2的光谱表征表明其含有黄素。将纯的重组FLbR - 2与大豆Lba和烟酰胺腺嘌呤二核苷酸（NADH）一起孵育，时间动力学速率表明重组FLbR - 2能有效地还原Lb3+。
