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 (34) 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.