title: Expression, Crystallization and Preliminary X-ray Diffraction Analyses of Med-ORF10 in the Biosynthetic Pathway of an Antitumor Antibiotic Medermycin.
abstract: 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 Å 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 Å resolution. These studies provide evidences for the functional Med-ORF10 protein in Streptomyces strains and facilitate our further investigation.
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Shasha
authors.initials: S
authors.lastname: Liu
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Yang
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Guo
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Yali
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Kashif Rafiq
authors.initials: KR
authors.lastname: Zahid
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Ke
authors.initials: K
authors.lastname: Liu
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Jinlin
authors.initials: J
authors.lastname: Liu
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Jihong
authors.initials: J
authors.lastname: Yang
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China.
authors.forename: Haobin
authors.initials: H
authors.lastname: Zhao
authors.affiliation: The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050091, People's Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: School of Life Sciences, State Key Laboratory of Microbial Technology, Shandong University-Helmholtz Institute of Biotechnology, Shandong University, Jinan, 250100, People's Republic of China. ayli@sdu.edu.cn.
authors.forename: Aiying
authors.initials: A
authors.lastname: Li
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People's Republic of China. qichao@mail.ccnu.edu.cn.
authors.forename: Chao
authors.initials: C
authors.lastname: Qi
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D009285:Naphthoquinones / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000235:genetics*; D014961:X-Ray Diffraction; D000095622:Benzoisochromanequinones
chemical_list: D000900:Anti-Bacterial Agents; D009285:Naphthoquinones; D011994:Recombinant Proteins; C013104:medermycin; D000095622:Benzoisochromanequinones
keywords: Crystallization; Expression and purification; Gene cloning; Med-ORF10; X-ray diffraction