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

查看全文 作　　者：ZHONG Gen [1,2]Shen;GUO Xiao [2]Fang;ZHANG Sheng [1]Hua;ZHEN Yong [1]Su 高影响力作者 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高影响力机构 出　　处：《Biomedical and Environmental Sciences》索引2011年第24卷第6期,共6页高影响力期刊 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°C,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：力达霉素 条件优化 载脂蛋白 生色团 反相高效液相色谱法 正交试验设计 最佳工艺条件 装配条件
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