Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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One-Step Purification of Melittin Derived from Apis mellifera Bee Venom

  • Received : 2016.08.22
  • Accepted : 2016.09.20
  • Published : 2017.01.28
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Abstract

The concern over the use of melittin in honey bee venom due to its adverse reaction caused by allergens such as phospholipase A2 ($PLA_2$) and hyaluronidase (HYA) has been an obstacle towards its usage. We developed a novel single-step method for melittin purification and the removal of $PLA_2$ and HYA. This study explores the influence of pH, buffer compositions, salt concentration, and types of cation-exchange chromatography resins on the recovery of melittin and the removal of both HYA and $PLA_2$. Melittin was readily purified with a strong cation-exchange resin at pH 6.0 with sodium phosphate buffer. It resulted in a recovery yield of melittin up to 93% (5.87 mg from a total of 6.32 mg of initial melittin in crude bee venom), which is higher than any previously reported studies on melittin purification. $PLA_2$ (99%) and HYA (96%) were also successfully removed. Our study generates a single-step purification method for melittin with a high removal rate of $PLA_2$ and HYA, enabling melittin to be fully utilized for its therapeutic purposes.

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References

  1. Moon DO, Park SY, Lee KJ, Heo MS, Kim KC, Kim MO, et al. 2007. Bee venom and melittin reduce proinflammatory mediators in lipopolysaccharide-stimulated BV2 microglia. Int. Immunopharmacol. 7: 1092-1101. https://doi.org/10.1016/j.intimp.2007.04.005
  2. Maulet Y, Brodbeck U, Fulpius BW. 1982. Purification from bee venom of melittin devoid of phospholipase A2 contamination. Anal. Biochem. 127: 61-67. https://doi.org/10.1016/0003-2697(82)90144-0
  3. Vila-Farres X, Giralt E, Vila J. 2012. Update of peptides with antibacterial activity. Curr. Med. Chem. 19: 6188-6198. https://doi.org/10.2174/0929867311209066188
  4. Wu R, Wang Q, Zheng Z, Zhao L, Shang Y, Wei X, et al. 2014. Design, characterization and expression of a novel hybrid peptides melittin (1-13)-LL37 (17-30). Mol. Biol. Rep. 41: 4163-4169. https://doi.org/10.1007/s11033-013-2900-0
  5. Stockwell VO, Duffy B. 2012. Use of antibiotics in plant agriculture. Rev. Sci. Tech. 31: 199-210. https://doi.org/10.20506/rst.31.1.2104
  6. Cichocka-Jarosz E. 2012. Hymenoptera venom allergy in humans. Folia Med. Cracov. 52: 43-60.
  7. Moreno M, Giralt E. 2015. Three valuable peptides from bee and wasp venoms for therapeutic and biotechnological use: melittin, apamin and mastropan. Toxins (Basel) 7: 1126-1150. https://doi.org/10.3390/toxins7041126
  8. WHO/IUIS. 2016. Allergen Nomenclature. Available from http://www.allergen.org. Accessed August 12, 2016.
  9. Zhu W, Wang B, Zhu X. 2002. Isolation and purification of BV I-2H from bee venom and analysis of its biological action. Chin. Sci. Bull. 47: 910-914. https://doi.org/10.1360/02tb9204
  10. Stenholm A, Lindgren H, Shaffie J. 2006. Comparison of amine-selective properties of weak and strong cationexchangers. J. Chromatogr. A 1128: 73-78. https://doi.org/10.1016/j.chroma.2006.06.042
  11. Schmidt M, Hafner M, Frech C. 2014. Modeling of salt and pH gradient elution in ion-exchange chromatography. J. Sep. Sci. 37: 5-13. https://doi.org/10.1002/jssc.201301007
  12. GE Healthcare. Ion Exchange Chromatography and Chromatofocusing: Principles and Methods. GE Healthcare Handbooks.
  13. Corporation WB. 1993. Worthington Enzyme manual. Available from http://www.worthington-biochem.com/PLA/assay.html. Accessed January 05, 2016.
  14. Sigma-Aldrich. Enzymatic Assay of Hyaluronidase. Available from http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-hyaluronidase.html. Accessed January 20, 2016.
  15. Hall T, Wilson JJ, Brownlee TJ, Swartling JR, Langan SE, Lambooy PK. 2015. Alkaline cation-exchange chromatography for the reduction of aggregate and a mis-formed disulfide variant in a bispecific antibody purification process. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 975: 1-8. https://doi.org/10.1016/j.jchromb.2014.11.002
  16. McDonald P, Tran B, Williams CR, Wong M, Zhao T, Kelley BD, Lester P. 2016. The rapid identification of elution conditions for therapeutic antibodies from cation-exchange chromatography resin using high-throughput screening. J. Chromatogr. A 1433: 66-74. https://doi.org/10.1016/j.chroma.2015.12.071
  17. Staby A, Jacobsen JH, Hansen RG, Bruus UK, Jensen IH. 2006. Comparison of chromatographic ion-exchange resins V. Strong and weak cation-exchange resins. J. Chromatogr. A 1118: 168-179. https://doi.org/10.1016/j.chroma.2006.03.116

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