The characterization of purified recombinant protein CRM197 as a tool to study diphtheria toxin

1Manoilov, KYu., 2Gorbatiuk, OB, 2Usenko, MO, 1Shatursky, OYa., 1Borisova, TA, 1Kolibo, DV
1O. V. Palladin Institute of Biochemistry of the NAS of Ukraine, Kyiv
2Institute of Molecular Biology and Genetics of the NAS of Ukraine, Kyiv; State Institute of Genetic and Regenerative Medicine of the NAMS of Ukraine, Kyiv
Dopov. Nac. akad. nauk Ukr. 2016, 9:124-133
https://doi.org/10.15407/dopovidi2016.09.124
Section: Biochemistry
Language: Ukrainian
Abstract: 

The purification of non-toxic diphtheria toxoid CRM197 expressed in Esherichia coli by metal-affinity chromatography following the enzymatic digestion of a bacterial cell wall and DNA allowed us to avoid the contamination by endogenous pore-forming proteins. It was shown that the fluorescein isothiocyanate-labeled samples of CRM197 are capable of binding and internalizing by mammalian cells Vero and L929. The introduction of CRM197 (2–20 nM) at positive voltages from the side of phosphatidylethanolamine-containing bilayer membrane (BLM ), were the toxoid was added, resulted in the creation of potential-dependent ionic channels with the conductance of 20 pS in the bathing solution of 1M KCl buffered at pH 4.8, as had been shown in classic studies of wild-type diphtheria toxin. The activation energies measured for the CRM197-created steady-state macroscopic current on one side of BLM in the solution of 1M KCl (pH 6.0) and the solution without membrane are equal to 2.967 ± 0.167 kcal/mol and 2.933 ± 0.115 kcal/mol, respectively, which suggests the formation of a transmembrane water pore by CRM197.

Keywords: black lipid membranes, CRM197, diphtheria toxin, membrane channels, toxoid
References: 
  1. Labyntsev A. J., Korotkevych N. V., Manoilov K. J., Kaberniuk A. A., Kolybo D. V., Komisarenko S. V. Russ. J. Bioorganic Chem., 2014, 40: 401–409. https://doi.org/10.1134/S1068162014040086
  2. Malito E., Bursulaya B., Chen C., Lo Surdo P., Picchianti M., Balducci E., Biancucci M., Brock A., Berti F., Bottomley M.J., Nissum M., Costantino P., Rappuoli R., Spraggon G. Proc. Natl. Acad. Sci. U.S.A., 2012, 109: 5229–5234. https://doi.org/10.1073/pnas.1201964109
  3. Donovan J.J., Simon M.I., Draper R.K., Montal M. Proc. Natl. Acad. Sci. U.S.A., 1981, 78: 172–176. https://doi.org/10.1073/pnas.78.1.172
  4. Gorbatiuk O. B., Tsapenko M. V., Pavlova M. V., Okunev O. V., Kordium V. A. Biopolym. Cell., 2012, 28: 141–148. https://doi.org/10.7124/bc.000041
  5. Basu A., Li X., Leong S. S. J. Appl. Microbiol. Biotechnol., 2011, 92: 241–251. https://doi.org/10.1007/s00253-011-3513-y
  6. Villaverde A. Carrió M. M. Biotechnol. Lett., 2003, 25: 1385–1395. https://doi.org/10.1023/A:1025024104862
  7. Gabliks J., Falconer M. J. Exp. Med., 1966, 123: 723–732. https://doi.org/10.1084/jem.123.4.723
  8. Mitamura T., Higashiyama S., Taniguchi N., Klagsbrun M., Mekada E. J. Biol. Chem., 1995, 270: 1015–1019. https://doi.org/10.1074/jbc.270.3.1015
  9. Moehring J. M., Moehring T. J. Infect. Immun., 1976, 13: 221–228.
  10. Gibson A. E., Noel R. J., Herlihy J. T., Ward W. F. Am. J. Physiol., 1989, 257: C182–184.
  11. Senzel L., Huynh P. D., Jakes K. S., Collier R. J., Finkelstein A. J. Gen. Physiol., 1998, 112: 317–324.https://doi.org/10.1085/jgp.112.3.317
  12. Kagan B. L., Reich K. A., Collier R. J. Biophys. J., 1984, 45: 102–104. https://doi.org/10.1016/S0006-3495(84)84126-0