Nozaki Seishiro, Tomioka Yoshihisa, Hishinuma Takanori, Inoue Masayuki, Nagumo Yoko, Tsuruta Lilian R, Hayashi Katsuhiro, Matsumoto Toshiki, Kato Yoshinori, Ishiwata Shunji, Itoh Kunihiko, Suzuki Toshio, Hirama Masahiro, Mizugaki Michinao
Department of Pharmaceutical Sciences, Tohoku University Hospital and Division of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Tohoku University, Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan.
J Biochem. 2002 May;131(5):729-38. doi: 10.1093/oxfordjournals.jbchem.a003158.
Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 +/- 0.3 microM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer polymerase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (DeltaIT(max)) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.
新制癌菌素（NCS）是首个被发现的具有含烯二炔发色团和载脂蛋白且二者以1:1复合物形式存在的抗肿瘤抗生素。在噬菌体展示载体pJuFo上设计并构建了用于在大肠杆菌中生产NCS载脂蛋白（脱辅基NCS）的人工基因文库。用小鼠抗脱辅基NCS单克隆抗体1C7D4富集表达前脱辅基NCS蛋白的重组噬菌体。成功克隆了大肠杆菌的脱辅基NCS基因（encsA），然后将其重新克隆到pRSET A载体中。在对带有组氨酸标签的脱辅基NCS蛋白进行纯化并用肠激酶切割后，使用BEACON 2000系统和GraphPad Prism软件通过监测总荧光强度和荧光偏振来研究重组蛋白与溴化乙锭（EtBr）的结合特性。在荧光偏振研究中，重组脱辅基NCS的解离常数为4.4±0.3微摩尔。这表明以EtBr作为发色团模拟物进行荧光偏振监测可能是一种用于表征重组脱辅基NCS与NCS发色团结合的简化方法。当使用两步法大引物聚合酶链反应通过定点诱变将脱辅基NCS上的苯丙氨酸78替换为色氨酸78时，尽管计算得出的总荧光强度最大变化（DeltaIT(max)）从113.9降至31.3，但在荧光偏振分析中观察到的脱辅基NCS突变体与EtBr的结合程度与野生型相同。这表明与野生型脱辅基NCS相比，F78W上结合的EtBr分子的环境可能发生了显著变化。这种荧光偏振和总荧光强度监测的组合将适用于确定和预测与靶蛋白结合或相关的配体状态。还使用这种重组脱辅基NCS制剂以及小牛胸腺组蛋白H1、H2A、H2B、H3和H4对组蛋白特异性蛋白水解活性进行了重新研究。重组脱辅基NCS不具有组蛋白蛋白酶的作用，因为在我们的实验条件下，在有和没有脱辅基NCS的孵育混合物之间未观察到明显差异。
