Suen W C, Gibson D T
Department of Microbiology, University of Iowa, Iowa City 52242.
Gene. 1994 May 27;143(1):67-71. doi: 10.1016/0378-1119(94)90606-8.
Pseudomonas sp. strain NCIB 9816-4 utilizes naphthalene dioxygenase (NDO), a multicomponent enzyme system, to initiate naphthalene degradation. The terminal component of NDO is an iron-sulfur protein (ISPNAP) with an alpha 2 beta 2 subunit composition. The structural genes encoding the alpha (nahAc) and beta (nahAd) subunits were cloned separately and together into expression vectors where transcription is under the control of the T7 promoter. The recombinant plasmids were transformed into Escherichia coli JM109[pGP1-2] and the synthesis of ISPNAP and its alpha and beta subunits was determined by SDS-PAGE. Low expression of nahAd was shown to be due to inefficient initiation of translation, but a sixfold increase in the amount of beta subunit synthesized was achieved in a coupled translation system. Inclusion bodies were found in all recombinants. Increased levels of soluble active proteins were obtained when E. coli JM109(DE3), used as the host strain for recombinant plasmid, was grown at 25 degrees C. ISPNAP from JM109(DE3)[pDTG121] was purified to homogeneity and shown to have the same properties as those determined for the enzyme purified from NCIB 9816-4. Active ISPNAP was also obtained by mixing cell extracts from separate strains that synthesized the alpha and beta subunits. The availability of large amounts of purified ISPNAP and its alpha and beta subunits will facilitate future studies on the mechanism of oxygen fixation by NDO.
假单胞菌属菌株NCIB 9816 - 4利用萘双加氧酶（NDO）（一种多组分酶系统）启动萘的降解。NDO的末端组分是一种具有α2β2亚基组成的铁硫蛋白（ISPNAP）。编码α（nahAc）和β（nahAd）亚基的结构基因被分别克隆，并一起克隆到转录受T7启动子控制的表达载体中。将重组质粒转化到大肠杆菌JM109[pGP1 - 2]中，通过SDS - PAGE测定ISPNAP及其α和β亚基的合成情况。结果表明，nahAd的低表达是由于翻译起始效率低下，但在偶联翻译系统中β亚基的合成量增加了六倍。在所有重组体中均发现了包涵体。当将大肠杆菌JM109(DE3)用作重组质粒的宿主菌株并在25℃下培养时，可获得更高水平的可溶性活性蛋白。从JM109(DE3)[pDTG121]中纯化得到的ISPNAP达到了均一性，并且显示出与从NCIB 9816 - 4中纯化得到的酶具有相同的性质。通过混合分别合成α和β亚基的不同菌株的细胞提取物，也获得了活性ISPNAP。大量纯化的ISPNAP及其α和β亚基的可得性将有助于未来对NDO氧固定机制的研究。
