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Expression, Crystallization and Preliminary X-ray Diffraction Analyses of Med-ORF10 in the Biosynthetic Pathway of an Antitumor Antibiotic Medermycin

The Protein Journal volume 34, pages 404–410 (2015)Cite this article

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.

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Abbreviations

MED:

Medermycin

ORF:

Open reading frame

BIQ:

Benzoisochromanequinones

ACT:

Actinorhodin

GRA:

Granaticin

E. coli :

Escherichia coli

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Acknowledgments

We are grateful to Prof. Ichinose Koji for the generous gift of plasmid pIK340. We would like to thank Aiping Dong for collecting the data and revising the manuscript. Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. This study was supported by the project of Hubei Key Laboratory of Genetic Regulation and Integrative Biology (GRIB201403) and the Fundamental Research Funds for the Central Universities (No. CCNU15A05036).Thanks for financial supports by the National Natural Science Foundation of China (Nos. 31500613, 31170050) and the Xinjiang production and construction corps key laboratory of protection and utilization of biological resources in Tarim Basin (No. BRZD1401).

Author Contributions

Yanli Liu, Shasha Liu and Ke Liu purified, crystallized the protein and conducted the statistics analyses of the diffraction data; Tingting Yang, Xiaoxia Guo and Yali Jiang cloned the gene; Jinlin Liu and Jihong Yang performed western blotting of Med-ORF10; Haobin Zhao and Yi Yang made the table and figures; Chao Qi and Aiying Li conceived of and designed the experiment; Yanli Liu, Aiying Li, Kashif Rafiq Zahid and Chao Qi wrote the paper.

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Affiliations

  1. Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, People’s Republic of China

    Yanli Liu, Shasha Liu, Tingting Yang, Xiaoxia Guo, Yali Jiang, Kashif Rafiq Zahid, Ke Liu, Jinlin Liu, Jihong Yang, Haobin Zhao & Chao Qi

  2. 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

    Aiying Li

  3. The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050091, People’s Republic of China

    Yi Yang

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  1. Yanli Liu
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  2. Shasha Liu
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  3. Tingting Yang
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  4. Xiaoxia Guo
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  5. Yali Jiang
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  6. Kashif Rafiq Zahid
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  7. Ke Liu
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  8. Jinlin Liu
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  9. Jihong Yang
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  10. Haobin Zhao
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  11. Yi Yang
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  12. Aiying Li
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  13. Chao Qi
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Correspondence to Aiying Li or Chao Qi.

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Cite this article

Liu, Y., Liu, S., Yang, T. et al. Expression, Crystallization and Preliminary X-ray Diffraction Analyses of Med-ORF10 in the Biosynthetic Pathway of an Antitumor Antibiotic Medermycin. Protein J 34, 404–410 (2015). https://doi.org/10.1007/s10930-015-9635-9

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  • DOI: https://doi.org/10.1007/s10930-015-9635-9

Keywords

  • Med-ORF10
  • Gene cloning
  • Expression and purification
  • Crystallization
  • X-ray diffraction