标题：Expression, Crystallization and Preliminary X-ray Diffraction Analyses of Med-ORF10 in the Biosynthetic Pathway of an Antitumor Antibiotic Medermycin
作者：Liu, Yanli; Liu, Shasha; Yang, Tingting; Guo, Xiaoxia; Jiang, Yali; Zahid, Kashif Rafiq; Liu, Ke; Liu, Jinlin; Yang, Jihong; Zhao, H 更多 作者机构：[Liu, Yanli; Liu, Shasha; Yang, Tingting; Guo, Xiaoxia; Jiang, Yali; Zahid, Kashif Rafiq; Liu, Ke; Liu, Jinlin; Yang, Jihong; Zhao, Haobin; Qi, Chao] 更多
通讯作者地址：[Li, AY]Shandong Univ, Shandong Univ Helmholtz Inst Biotechnol, State Key Lab Microbial Technol, Sch Life Sci, Jinan 250100, Peoples R China.
关键词：Med-ORF10; Gene cloning; Expression and purification; Crystallization;; X-ray diffraction
摘要：Medermycin, as a prominent member of benzoisochromanequinones, possesses strong antitumor activity and is biosynthesized under the control of a 29-ORF-containing biosynthetic gene cluster. Most of ORFs in this gene cluster have not been characterized, including a small protein encoding gene med-ORF10, proposed to play a regulatory role in biosynthesis of medermycin in an unknown mode. In this study, we reported the expression, protein preparation, crystallization and preliminary X-ray diffraction analyses of Med-ORF10 of the wild type Streptomyces strain. Firstly, we cloned and overexpressed med-ORF10 in Escherichia coli and purified the protein with 98 % purity and 3 mg/L yield. Then, we crystallized the protein at concentration of 20 mg/mL in condition 22 % PEG 3350, 0.2 M magnesium formate and collected the data at 1.78 resolution. Finally, we detected the expression of Med-ORF10 in Streptomyces by western blotting. In conclusion, this study confirmed the expression of Med-ORF10 protein in the wild-type strain of Streptomyces AM-7161 and collected the X-ray diffraction data of Med-ORF10 crystal at 1.78 resolution. These studies provide evidences for the functional Med-ORF10 protein in Streptomyces strains and facilitate our further investigation.