Optimization of the Assembly Efficiency for Lidamycin Chromophore Bound to its Apoprotein:A Case Study using Orthogonal Array  

作　　者：ZHONG Gen Shen GUO Xiao Fang ZHANG Sheng Hua ZHEN Yong Su 

机构地区：[1]Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences ＆ Peking Union Medical College, Beijing 100050, China [2]Xinxiang Medical University, Xinxiang 453003, Henan, China

出　　处：《Biomedical and Environmental Sciences》2011年第6期602-607,共6页生物医学与环境科学（英文版）

基　　金：supported by grants from "Significant new drug development" Science and Technology Major Projects of China (2009ZX09301-003; 2009ZX09401-005; 2010ZX09401-407)

摘　　要：Objective Lidamycin （LDM） can be dissociated to an apoprotein （LDP） and an active enediyne chromophore （AE）. The detached AE can reassemble with its LDP-containing fusion protein to endow the latter with potent antitumor activity. However, the reassembly of AE with LDP is affected by several factors. Our aim was to optimize the assembly efficiency of the AE with a LDP-containing fusion protein and investigate the influence of several factors on the assembly efficacy. Methods A method based on RP-HPLC was developed to analyze the assembly rate, and an orthogonal experimental design L9 （3^4） was used to investigate the effects of temperature, assembly time, pH and molecular ratio of LDP-containing fusion protein to AE on the assembly rate. Furthermore, the determined optimum conditions for the assembly rate of the LDP-containing fusion protein with AE were applied and evaluated. Results A calibration curve based on the LDM micromolar concentration against the peak-area of AE by HPLC was obtained. The order in which individual factors in the orthogonal experiment affected the assembly rate were temperature〉time〉pH〉molar ratio of AE to protein and all were statistically significant （P〈0.01）. The optimal assembly conditions were temperature at 10 ℃, time of 12 h, pH 7.0, and the molar ratio of AE： protein of 5：1. The assembly rate of AE with a LDP-containing fusion protein was improved by 23% after condition optimization. Conclusion The assembly rate of chromophore of lidamycin with its LDP-containing fusion protein was improved after condition optimization by orthogonal design, and the optimal conditions described herein should prove useful for the development of this type of LDP-containing fusion protein.Objective Lidamycin （LDM） can be dissociated to an apoprotein （LDP） and an active enediyne chromophore （AE）. The detached AE can reassemble with its LDP-containing fusion protein to endow the latter with potent antitumor activity. However, the reassembly of AE with LDP is affected by several factors. Our aim was to optimize the assembly efficiency of the AE with a LDP-containing fusion protein and investigate the influence of several factors on the assembly efficacy. Methods A method based on RP-HPLC was developed to analyze the assembly rate, and an orthogonal experimental design L9 （3^4） was used to investigate the effects of temperature, assembly time, pH and molecular ratio of LDP-containing fusion protein to AE on the assembly rate. Furthermore, the determined optimum conditions for the assembly rate of the LDP-containing fusion protein with AE were applied and evaluated. Results A calibration curve based on the LDM micromolar concentration against the peak-area of AE by HPLC was obtained. The order in which individual factors in the orthogonal experiment affected the assembly rate were temperature〉time〉pH〉molar ratio of AE to protein and all were statistically significant （P〈0.01）. The optimal assembly conditions were temperature at 10 ℃, time of 12 h, pH 7.0, and the molar ratio of AE： protein of 5：1. The assembly rate of AE with a LDP-containing fusion protein was improved by 23% after condition optimization. Conclusion The assembly rate of chromophore of lidamycin with its LDP-containing fusion protein was improved after condition optimization by orthogonal design, and the optimal conditions described herein should prove useful for the development of this type of LDP-containing fusion protein.

关 键 词：LIDAMYCIN CHROMOPHORE HPLC Orthogonal array Assembly rate Lidamycin CHROMOPHORE HPLC Orthogonal array Assembly rate 

分 类 号：O631.24[理学—高分子化学] Q933[理学—化学]