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Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities

  • Applied genetics and molecular biotechnology
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Abstract

The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 μg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC50 = 13–15 μg/ml).

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Acknowledgments

We gratefully acknowledge Dr. Thomas Ruppert (Head of Mass Spectrometry, ZMBH, Heidelberg University, Germany) for carrying out MS measurements. Thanks to Theodor C. H. Cole for proofreading and for valuable comments. R. A. Aleinein and R. Hamoud are grateful to the Syrian Ministry of Higher Education and Tishreen University, Syria, for financial support.

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Authors and Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120, Heidelberg, Germany

    Rasha Abou Aleinein, Razan Hamoud, Holger Schäfer & Michael Wink

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  1. Rasha Abou Aleinein
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  2. Razan Hamoud
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  3. Holger Schäfer
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  4. Michael Wink
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Correspondence to Michael Wink.

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Aleinein, R.A., Hamoud, R., Schäfer, H. et al. Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities. Appl Microbiol Biotechnol 97, 3535–3543 (2013). https://doi.org/10.1007/s00253-012-4441-1

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  • DOI: https://doi.org/10.1007/s00253-012-4441-1

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