Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli
Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli
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大肠杆菌中表达的果蝇酚氧化酶的特性
DOI：
10.1016/j.dci.2011.11.005
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发表时间：
2012-04-01
影响因子：
2.9
通讯作者：
Ling, Erjun
中科院分区：
文献类型：
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作者：
Li, Xuquan;Ma, Miaolian;Ling, Erjun
昆虫酚氧化酶原（PPOs）是一类重要的先天免疫蛋白。尽管已经有大量研究涉及PPO激活级联，但PPO家族蛋白的详细生化行为仍有待明确建立。这主要是由于难以获得足够数量的PPO蛋白进行全面表征。在本研究中，我们在大肠杆菌中表达了三个黑腹果蝇PPO基因，并广泛评估了获得可溶性蛋白的表达条件。通过调控PPO转化的大肠杆菌细胞的表达条件，特别是培养温度，我们能够获得大量的可溶性重组PPO蛋白。在PPO诱导过程中，在培养基中加入额外的Cu2+，或者直接与纯化的rPPO制剂混合，都是产生Cu2+相关前酶所必需的。Cu2+相关的PPOs在被乙醇或氯化十六烷基吡啶或AMM1（一种含有天然丝氨酸蛋白酶的蛹蛋白组分）激活后显示出明显的酶活性。纯化个体果蝇rPPOs与Cu2+关联的剂量反应表明，果蝇rPPO1和rPPO3对Cu2+的亲和力相对高于rPPO2。然而，令人惊讶的是，高浓度的Cu2+ (2 mM)完全抑制PPO活性。当多巴胺或左旋多巴为底物时，每个rPPO都有相似的活性。然而，如果以l -酪氨酸作为底物，单独的rPPO1具有非常高的活性。经乙醇或2-丙醇活化后，3种rPPOs的Km和Vmax变化规律如下：rPPO2 < rPPO3 < rPP01。经乙醇活化后，各rPPO的Km和Vmax均低于2-丙醇。由于传统纯化方法难以获得功能性PPOs，本研究建立的方法将有助于制备活性昆虫重组PPOs，为今后研究PPO的性质和功能提供依据。(C) 2011 Elsevier Ltd.版权所有。
Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for comprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in Escherichia coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of expression conditions, particularly the culture temperature of PPO-transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. Additional Cu2+, either added into the culture medium during PPO induction or directly mixed with the purified rPPO preparations, was necessary to produce Cu2+ associated proenzymes. Cu2+ associated PPOs showed obvious enzyme activities after activation by either ethanol or cetylpyridinium chloride, or by AMM1 (a pupal protein fraction containing native serine proteases for PPO activation). Dose responses for association of individual purified Drosophila rPPOs with Cu2+ showed that Drosophila rPPO1 and rPPO3 had relatively higher affinity for Cu2+ than rPPO2 did. Surprisingly, however, high concentration of Cu2+ (2 mM) completely inhibited PPO activity. Each rPPO had similar activity when dopamine or L-DOPA was the substrate. However, rPPO1 alone had very high activity if L-tyrosine was used as a substrate. After activation by ethanol or 2-propanol, Km and Vmax of the three rPPOs changed as shown in the following: rPPO2 < rPPO3 < rPP01. If activated by ethanol, the Km and Vmax of each rPPO were lower than by 2-propanol. Due to the difficulty in obtaining functional PPOs via traditional purification methods, the method established in this study will be helpful to produce active insect recombinant PPOs for the study of PPO properties and functions in the future. (C) 2011 Elsevier Ltd. All rights reserved.