Expression features of hybrid gene of frog antimicrobial peptide and staphylococcal bacteriophage K endolysin in bacteria Еsсherichia coli

Authors

  • Natalia V. Sovgir Belarusian State University, Nezavisimosti avenue, 4, 220030, Minsk, Republic of Belarus
  • Sergey G. Golenchenko Belarusian State University, Nezavisimosti avenue, 4, 220030, Minsk, Republic of Belarus
  • Vladimir A. Prakulevich Belarusian State University, Nezavisimosti avenue, 4, 220030, Minsk, Republic of Belarus

Keywords:

antimicrobial peptides, esculentin, staphylococcal bacteriophage K endolysin, fusion protein, antistaphylococcal activity, CFU (colony forming unit)

Abstract

The aim of this study was cloning and expression analysis of hybrid gene consisted of frog (Rana esculenta L., 1758) antimicrobial peptide esculentin gene and the Myoviridae family staphylococcal bacteriophage K endolysin gene in Е. coli cells. Primers to fuse esculentin and staphylococcal bacteriophage K endolysin genes were designed to obtain the chimeric protein with His-tag at the C-terminus. The hybrid gene was cloned in E. coli cells and sequenced. The subsequent analysis of gene expression revealed the intracellular accumulation of fusion protein esculentin-C / endolysin KHis. Recombinant protein accumulated in E. coli cells predominantly in the insoluble form, but some of protein molecules remained soluble and exhibited antistaphylococcal activity. Applications: biotechnology, molecular biology, pharmaceutics.

Author Biographies

  • Natalia V. Sovgir, Belarusian State University, Nezavisimosti avenue, 4, 220030, Minsk, Republic of Belarus

    assistant at the department of microbiology, faculty of biology.

  • Sergey G. Golenchenko, Belarusian State University, Nezavisimosti avenue, 4, 220030, Minsk, Republic of Belarus

    researcher at the department of microbiology, faculty of biology.

  • Vladimir A. Prakulevich, Belarusian State University, Nezavisimosti avenue, 4, 220030, Minsk, Republic of Belarus

    doctor of science (biology), full professor; head of the department of microbiology, faculty of biology.

References

  1. Anthony A. A., Adekunle C. F., Gulshan S., et al. Antibiotic resistant superbugs: assessment of the interrelationship of occurrence in clinical settings and environmental niches. Molecules. 2017. Vol. 22, No. 1. Р. 29–45.
  2. Cappiello F., Di Grazia A., Segev-Zarko L. A., et al. Esculentin-1a-derived peptides promote clearance of Pseudomonas aeruginosa Internalized in bronchial cells of cystic fibrosis patients and lung cell migration: biochemical properties and a plausible mode of action. Antimicrob. Agents Chemother. 2016. Vol. 60, No. 12. P. 7252–7262.
  3. Omardien S., Brul S., Zaat S. A. J. Antimicrobial activity of cationic antimicrobial peptides against gram-positives: current progress made in understanding the mode of action and the response of bacteria. Front. Cell Dev. Biol. 2016. Vol. 4, No. 111. P. 1–16.
  4. Ponti D., Mignogna G., Mangoni M. L., et al. Expression and activity of cyclic and linear analogues of esculentin-1, an antimicrobial peptide from amphibian skin. Eur. J. Biochem. 1999. Vol. 263, No. 3. P. 921–927.
  5. Sovgir N. V., Prokulevich V. A. Cloning and expression of antimicrobial peptide esculentin-1b gene (Rana esculenta) in Escherichia coli cells. Tr. BGU. Ser.: Fiziol., biokhim. i mol. osnovy funkts. biosist. 2011. Vol. 8 : in 2 parts. Part 1. P. 70–75 (in Russ.).
  6. Golenchenko S. G., Prokulevich V. A. Synthesis and expression of staphilococcal phage lysin gene in E. coli. Tr. BGU. Ser.: Fiziol., biokhim. i mol. osnovy funkts. biosist. 2012. Vol. 7, part 1. P. 199–204 (in Russ.).
  7. Horgan M., O’Flynn G., Garry J., et al. Phage Lysin LysK can be truncated to its chap domain and retain lytic activity against live antibiotic-resistant Staphylococci. Appl. Environ. Microbiol. 2009. Vol. 75, No. 3. P. 872–874.
  8. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 1986. Vol. 189. P. 113–130.
  9. McPherson M. J., Moller S. G. PCR. Oxford, 2000. 10. OligoAnalyzer tool [Electronic resource]. URL: http://eu.idtdna.com/calc/analyzer (date of access: 15.02.2015).
  10. Maniatis T., Frich E., Sembruk Dzh. Metody geneticheskoi inzhenerii. Molekulyarnoe klonirovanie [Genetic engineering methods. Molecular cloning]. Moscow, 1984 (in Russ.).
  11. pET Manual system [Electronic resource]. 2005. URL: http://kirschner.med.harvard.edu/files/protocols/Novagen_petsystem. pdf/ (date of access: 02.06.2015) .
  12. Lysak V. V., Zheldakova R. A. Mikrobiologiya : metod. rekomendatsii k lab. zanyatiyam, kontrol’ samostoyat. raboty studentov [Microbiology : guidelines for laboratory work, control of student’s individual work]. Minsk, 2002 (in Russ.).
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970. Vol. 227. P. 680–685.
  14. ProtParam tool [Electronic resource]. Swiss Inst. Bioinform. 2014. URL: http://web.expasy.org/protparam/ (date of access: 17.02.2016) .
  15. Singh S. M., Panda A. K. Solubilization and refolding of bacterial inclusion body proteins. J. Biosci. Bioeng. 2005. Vol. 99. No. 4. Р. 303–310.

Downloads

Published

2017-12-03

Issue

No. 1 (2017): Journal of the Belarusian State University. Biology

Section

Genetics, Microbiology and Molecular Biology

License

The authors who are published in this journal agree to the following:

  1. The authors retain copyright on the work and provide the journal with the right of first publication of the work on condition of license Creative Commons Attribution-NonCommercial. 4.0 International (CC BY-NC 4.0).
  2. The authors retain the right to enter into certain contractual agreements relating to the non-exclusive distribution of the published version of the work (e.g. post it on the institutional repository, publication in the book), with the reference to its original publication in this journal.
  3. The authors have the right to post their work on the Internet (e.g. on the institutional store or personal website) prior to and during the review process, conducted by the journal, as this may lead to a productive discussion and a large number of references to this work. (See The Effect of Open Access.)

How to Cite

Sovgir, N. V., Golenchenko, S. G., & Prakulevich, V. A. (2017). Expression features of hybrid gene of frog antimicrobial peptide and staphylococcal bacteriophage K endolysin in bacteria Еsсherichia coli. Experimental Biology and Biotechnology, 1, 62-70. https://journals.bsu.by/index.php/biology/article/view/2430