PRODUCTION OF RECOMBINANT PROTEIN CRM197 IN ESCHERICHIA COLI

Cover Page


Cite item

Full Text

Abstract

The CRM197 is a non-toxic mutant of diphtheria toxin having a single amino acid substitution of a glycine for a glutamic acid in position 52. Being naturally nontoxic, CRM197 is a promising adjuvant and ideal carrier protein for conjugate vaccines. Typically, production of diphtheria toxin and some of the non-toxic proteins are carried out by Corynebacterium diphtheriae. Production of recombinant protein CRM197 in Escherichia coli is advantageous. It is simple, cheap and permits production of the target protein in a short time using a non-pathogenic microorganism. In this study patented high-yield-producing E. coli strain was used. As a part of the study the following steps were taken: protocol adjustment for induction of crm197 gene, production and purification of recombinant CRM197 by ion-exchange, hydrophobic and gel-filtration chromatography. The purity of the final preparation reached 97%.

About the authors

I. V. Dukhovlinov

197110, Russian Federation, St. Petersburg, Pudozhskaya str., 7, Research Institute of Highly Pure Biopreparations.

Author for correspondence.
Email: dukhovlinov@gmail.com

PhD (Biology), Head of the Laboratory of Genetic Engineering of Vaccines, Research Institute of Highly Pure Biopreparations, St. Petersburg, Russian Federation; 

Россия

E. A. Fedorova

197110, Russian Federation, St. Petersburg, Pudozhskaya str., 7, Research Institute of Highly Pure Biopreparations.

Email: fake@neicon.ru

Researcher, Laboratory of Genetic Engineering of Vaccines, Research Institute of Highly Pure Biopreparations, St. Petersburg, Russian Federation; 

Россия

E. G. Bogomolova

197110, Russian Federation, St. Petersburg, Pudozhskaya str., 7, Research Institute of Highly Pure Biopreparations.

Email: fake@neicon.ru

Junior Researcher, Laboratory of Genetic Engineering of Vaccines, Research Institute of Highly Pure Biopreparations, St. Petersburg, Russian Federation; 

Россия

O. A. Dobrovolskaya

197110, Russian Federation, St. Petersburg, Pudozhskaya str., 7, Research Institute of Highly Pure Biopreparations.

Email: fake@neicon.ru

Junior Researcher, Laboratory of Genetic Engineering of Vaccines, Research Institute of Highly Pure Biopreparations, St. Petersburg, Russian Federation; 

Россия

E. N. Chernyaeva

197110, Russian Federation, St. Petersburg, Pudozhskaya str., 7, Research Institute of Highly Pure Biopreparations.

Email: fake@neicon.ru

PhD (Biology), Senior Researcher, Laboratory of Genetic Engineering of Vaccines, Research Institute of Highly Pure Biopreparations, St. Petersburg, Russian Federation; 

Россия

R. I. Al-Shekhadat

197110, Russian Federation, St. Petersburg, Pudozhskaya str., 7, Research Institute of Highly Pure Biopreparations.

Email: fake@neicon.ru

PhD (Biology), The Deputy Head, Laboratory of Genetic Engineering of Vaccines, Research Institute of Highly Pure Biopreparations, St. Petersburg, Russian Federation; 

Россия

A. S. Simbirtsev

97110, Россия, Санкт-Петербург, ул. Пудожская, 7, ФГУП ГосНИИ ОЧБ ФМБА России.

Email: fake@neicon.ru

PhD, MD (Medicine), Professor, Director of the Research Institute of Highly Pure Biopreparations, St. Petersburg, Russian Federation. 

Россия

References

  1. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, vol. 72, pp. 248–254.
  2. Bromuro C., Romano M., Chiani P., Berti F., Tontini M., Proietti D., Mori E., Torosantucci A., Costantino P., Rappuoli R., Cassone A. Beta-glucan-CRM197 conjugates as candidates antifungal vaccines. Vaccine, 2010, vol. 28, no. 14, pp. 2615–2623.
  3. Bryant K.A., Block S.L., Baker S.A., Gruber W.C., Scott D.A. PCV13 Infant Study Group. Safety and immunogenicity of a 13-valent pneumococcal conjugate vaccine. Pediatrics, 2010, vol. 125, no. 5, pp. 866–875.
  4. Dagan R., Poolman J., Siegrist C.A. Glycoconjugate vaccines and immune interference: a review. Vaccine, 2010, vol. 28, no. 34, pp. 5513–5523.
  5. DeWalt B.W., Murphy J.C., Fox G.E., Willson R.C. Compaction agent clarification of microbial lysates. Protein Expr. Purif., 2003, vol. 28, no. 2, pp. 220–223.
  6. Gill C.J., Baxter R., Anemona A., Ciavarro G., Dull P. Persistence of immune responses after a single dose of Novartis meningococcal serogroup A, C, W-135 and Y CRM-197 conjugate vaccine (Menveo®) or Menactra® among healthy adolescents. Hum. Vaccin., 2010, vol. 6, no. 11, pp. 881–887.
  7. Hwang K.W. Haemophilus influenza type b (Hib) vaccine and its carrier proteins. Arch. Pharm. Res., 2010, vol. 33, no. 6, pp. 793–795.
  8. Kunami N., Yotsumoto F., Ishitsuka K., Fukami T., Odawara T., Manabe S., Ishikawa T., Tamura K., Kuroki M., Miyamoto S. Antitumor effects of CRM197, a specific inhibitor of HB-EGF, in T-cell acute lymphoblastic leukemia. Anticancer Res., 2011, vol. 31, no. 7, pp. 2483–2488.
  9. Kuo Y.C., Chung C.Y. Transcytosis of CRM197-grafted polybutylcyanoacrylate nanoparticles for delivering zidovudine across human brain-microvascular endothelial cells. Colloids Surf. B: Biointerfaces, 2012, vol. 91, pp. 242–249.
  10. LaemmliU.K.CleavageofstructuralproteinsduringtheassemblyoftheheadofbacteriophageT4.Nature,1970,vol.227,no.5259, pp. 680–685.
  11. LekaO.,ValleseF.,PirazziniM.,BertoP.,MontecuccoC.,ZanottiG.DiphtheriatoxinconformationalswitchingatacidicpH. FEBS J., 2014, vol. 281, no. 9, pp. 2115–2122.
  12. Li M., Su Z.G., Janson J.C. In vitro protein refolding by chromatographic procedures. Protein Expr. Purif., 2004, vol. 33, no. 1, pp. 1–10.
  13. Park K. Targeted delivery to monocytes. J. Control. Release, 2012, vol. 158, no. 1, p. 1.
  14. RivettiS.,LauriolaM.,VoltattorniM.,BianchiniM.,MartiniD.,CeccarelliC.,PalmieriA.,MatteiG.,FranchiM.,UgoliniG., Rosati G., Montroni I., Taffurelli M., Solmi R. Gene expression profile of human colon cancer cells treated with cross-reacting material 197, a diphtheria toxin non-toxic mutant. Int. J. Immunopathol. Pharmacol., 2011, vol. 24, no. 3, pp. 639–649.
  15. Safari D., Dekker H.A., De Jong B., Rijkers G.T., Kamerling J.P., Snippe H. Antibodyand cell-mediated immune responses to a synthetic oligosaccharide conjugate vaccine after booster immunization. Vaccine, 2011, vol. 29, no. 38, pp. 6498–6504.
  16. Schenk G.J., Haasnoot P.C., Centlivre M., Legrand N., Rip J., De Boer A.G., Berkhout B. Efficient CRM197-mediated drug targeting to monocytes. J. Control. Release, 2012, vol. 158, no. 1, pp. 139–147.
  17. Shinefield H.R. Overview of the development and current use of CRM(197) conjugate vaccines for pediatric use. Vaccine, 2010, vol. 28, no. 27, pp. 4335–4339.
  18. SkinnerJ.M.,IndrawatiL.,CannonJ.,BlueJ.,WintersM.,MacnairJ.,PujarN.,MangerW.,ZhangY.,AntonelloJ.,ShiverJ., Caulfield M., Heinrichs J.H. Pre-clinical evaluation of a 15-valent pneumococcal conjugate vaccine (PCV15-CRM197) in an infant-rhesus monkey immunogenicity model. Vaccine, 2011, vol. 29, no. 48, pp. 8870–8876.
  19. Stefan A., Conti M., Rubboli D., Ravagli L., Presta E., Hochkoeppler A. Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli. J. Biotechnol., 2010, vol. 156, no. 4, pp. 245–252.
  20. Studier F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif., 2005, vol. 41, no. 1, pp. 207–234.
  21. TangX.H.,DengS.,LiM.,LuM.S.Theanti-tumoreffectofcross-reactingmaterial197,aninhibitorofheparin-bindingEGFlike growth factor, in human resistant ovarian cancer. Biochem. Biophys. Res. Commun., 2012, vol. 422, no. 4, pp. 676–680.
  22. Uchida T., Pappenheimer A.M.Jr., Greany R. Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J. Biol. Chem., 1973, vol. 248, no. 11, pp. 3838–3844.
  23. Van den Biggelaar A.H., Pomat W., Bosco A., Phuanukoonnon S., Devitt C.J., Nadal-Sims M.A., Siba P.M., Richmond P.C., Lehmann D., Holt P.G. Pneumococcal conjugate vaccination at birth in a high-risk setting: no evidence for neonatal T-cell tolerance. Vaccine, 2011, vol. 29, no. 33, pp. 5414–5420.
  24. Yotsumoto F., Oki E., Tokunaga E., Maehara Y., Kuroki M., Miyamoto S. HB-EGF orchestrates the complex signals involved in triple-negative and trastuzumab-resistant breast cancer. Int. J. Cancer, 2010, vol. 127, no. 11, pp. 2707–2717.
  25. ZhangH.L.,YuanC.,ZhangD.M.,ShiH.S.,LiM.,LuoZ.C.,WanY.,LuL.,LuoS.T.,YangL.Anovelcombinedconjugatevaccine: enhanced immunogenicity of bFGF with CRM197 as a carrier protein. Mol. Med. Rep., 2011, vol. 4, no. 5, pp. 857–863.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download

Copyright (c) 2015 Dukhovlinov I.V., Fedorova E.A., Bogomolova E.G., Dobrovolskaya O.A., Chernyaeva E.N., Al-Shekhadat R.I., Simbirtsev A.S.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
Регистрационный номер и дата принятия решения о регистрации СМИ: серия ПИ № ФС 77 - 64788 от 02.02.2016.


This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies