High-level Expression of Tetanus Toxin Fragment C in Escherichia coli

Document Type: Original Articles

Authors

Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute, Agricultural Research, Education, and Extension Organization, Karaj, Iran

Abstract

Fragment C is the C-terminal domain of the heavy chain of tetanus toxin that can promote the immune response against the lethal dose of this toxin. Therefore, this portion can be considered as a candidate vaccine against tetanus infection, which occurs by Clostridium tetani. The present study aimed to compare the expression of tetanus toxin fragment C in Escherichia coli  BL21 (DE3) pLysS cells having a high tolerance to toxins between two different expression vectors, namely pET22b and pET28a, using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. After DNA extraction from Harvard CN49205 strain of C. tetani, the gene of interest was amplified using polymerase chain reaction, and then sequenced and cloned into the expression vectors of pET22b and pET28a, transformed into competent BL21 (DE3) pLysS cells, and finally expressed using an optimized protocol. The cells were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at four different incubation temperatures (i.e., 37, 33, 30, and 25 °C) and three different incubation times (i.e., 1, 2, and 3 h). Although the SDS-PAGE and western blot analyses confirmed the expression of the recombinant fragment C (r-fragment C) ligated into both of the expression vectors, pET28a showed a higher r-fragment C expression level than the other vector (38.66 mg/L versus 32.33 mg/L, P<0.05). An optimal expression condition was acquired 3 h after 1 mM IPTG induction at 25 °C. The results demonstrated that E. coli BL21 (DE3) pLysS as an expression host in combination with pET-28a as an expression vector was a more compatible expression system to express the fragment C of tetanus toxin, compared to E. coli BL21 (DE3) pLysS/pET-22b expression system. Overall, these results may represent an opportunity to improve the expression system for the production of tetanus toxin vaccine using recombinant protein strategy.

Keywords

Main Subjects


Article Title [French]

La forte expression du fragment C de la toxine tétanique dans Escherichia coli

Abstract [French]

Le fragment C'est le domaine C-terminal de la chaîne lourde de la toxine tétanique. Ce dernier favorisé la réponse immunitaire contre la dose létale de cette toxine et peut donc être considéré comme un candidat potentiel pour le vaccin contre l'infection tétanique causée par Clostridium tetani. Dans cette étude, l'expression du fragment C de la toxine tétanique dans des cellules BL21 (DE3) pLysS d'E. coli ayant une tolérance élevée aux toxines a été comparée en utilisant deux vecteurs d'expression différents, pET22b et pET28a, par SDS-PAGE et western blot. Après l’extraction de l'ADN de la souche Harvard CN49205 de C. tetani, le gène d'intérêt a été amplifié par PCR, séquencé, cloné dans les vecteurs d'expression, pET22b et pET28a, transformé dans des cellules compétentes BL21 (DE3) pLysS et enfin exprimé selon un protocole optimisé. Les cellules ont été induites avec IPTG à quatre températures d'incubation différentes (37, 33, 30 et 25° C) et trois temps d'incubation différents (1 à 3 h). Bien que l'expression du fragment recombinant C (fragment C) ligaturé dans les deux vecteurs d'expression ait été confirmée par les analyses SDS-PAGE et western blot, pET28a a montré une expression plus élevée du fragment r C comparé à pET22b (38,66 mg / l contre 32,33 mg / l, p <0,5). La condition optimale d'expression a été obtenueà 5° C, IPTG 1 mM, et 3 h après l’induction de l’IPTG. Ces résultats ont démontré que l’hôte d'expression E. coli BL21 (DE3) pLysS en combinaison avec le vecteur deux vecteurs d'expression ait été confirmée par les analyses SDS-PAGE et western blot, pET28a a montré une expression plus élevée du fragment r C comparé à pET22b (38,66 mg / l contre 32,33 mg / l, p <0,5). La condition optimale d'expression a été obtenueà 5° C, IPTG 1 mM, et 3 h après l’induction de l’IPTG. Ces résultats ont démontré que l’hôte d'expression E. coli BL21(DE3) pLysS en combinaison avec le vecteur d'expression pET-28a représentait le système d'expression le plus compatible pour exprimer le fragment C de la toxine tétanique. Dans l’ensemble, ces résultats montrent qu’il est possible d’améliorer la production de vaccin contre la toxine tétanique à base de protéines recombinantes en optimisant leur système d'expression.

Keywords [French]

  • Clostridium tetani
  • Fragment C
  • vecteur d'expression pET22b
  • vecteur d'expression de pET28a
  • E. coli BL21 (DE3) pLysS
Bahreini, E., Aghaiypour, K., Abbasalipourkabir, R., Goodarzi, M.T., Saidijam, M., Safavieh, S.S., 2014. An optimized protocol for overproduction of recombinant protein expression in Escherichia coli. Prep Biochem Biotechnol 44, 510-528.

Bruggemann, H., Gottschalk, G., 2004. Insights in metabolism and toxin production from the complete genome sequence of Clostridium tetani. Anaerobe 10, 53-68.

Carlton, E., Teng, Q., Federici, T., Yang, J., Riley, J., Boulis, N.M., 2008. Fusion of the tetanus toxin C fragment binding domain and Bcl-xL for protection of peripheral nerve neurons. Neurosurgery 63, 1175-1182; discussion 1182-1174.

Charles, I.G., Rodgers, B.C., Makoff, A.J., Chatfield, S.N., Slater, D.E., Fairweather, N.F., 1991. Synthesis of tetanus toxin fragment C in insect cells by use of a baculovirus expression system. Infect Immun 59, 1627-1632.

Chatfield, S.N., Fairweather, N., Charles, I., Pickard, D., Levine, M., Hone, D., et al., 1992. Construction of a genetically defined Salmonella typhi Ty2 aroA, aroC mutant for the engineering of a candidate oral typhoid-tetanus vaccine. Vaccine 10, 53-60.

Dubendorf, J.W., Studier, F.W., 1991. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology 219, 45-59.

Fairweather, N.F., Lyness, V.A., 1986. The complete nucleotide sequence of tetanus toxin. Nucleic Acids Res 14, 7809-7812.

Fairweather, N.F., Lyness, V.A., Pickard, D.J., Allen, G., Thomson, R.O., 1986. Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli. J Bacteriol 165, 21-27.

Francis, J.W., Figueiredo, D., vanderSpek, J.C., Ayala, L.M., Kim, Y.S., Remington, M.P., et al., 2004. A survival motor neuron:tetanus toxin fragment C fusion protein for the targeted delivery of SMN protein to neurons. Brain Res 995, 84-96.

Gerday, C., Aittaleb, M., Bentahir, M., Chessa, J.-P., Claverie, P., Collins, T., et al., 1990. Cold-adapted enzymes: from fundamentals to biotechnology. Trends in Biotechnology 18, 103-107.

Gil, C., Chaib-Oukadour, I., Blasi, J., Aguilera, J., 2001. HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction. Biochem J 356, 97-103.

Goff, S.A., Goldberg, A.L., Production of abnormal proteins in E. coli stimulates transcription of ion and other heat shock genes. Cell 41, 587-595.

Grodberg, J., Dunn, J.J., 1988. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. Journal of Bacteriology 170, 1245-1253.

Halpern, J.L., Habig, W.H., Neale, E.A., Stibitz, S., 1990. Cloning and expression of functional fragment C of tetanus toxin. Infect Immun 58, 1004-1009.

Hannig, G., Makrides, S.C., 1998. Strategies for optimizing heterologous protein expression in Escherichia coli. Trends in Biotechnology 16, 54-60.

Helting, T.B., Nau, H.H., 1984. analysis of the immune response to papain digestion products of tetanus toxin. Acta Pathologica Microbiologica Scandinavica Series C: Immunology 92C, 59-63.

Helting, T.B., Zwisler, O., 1977. Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments. J Biol Chem 252, 187-193.

Helting, T.B., Zwisler, O., Wiegandt, H., 1977. Structure of tetanus toxin. II. Toxin binding to ganglioside. J Biol Chem 252, 194-198.

Herreros, J., Lalli, G., Schiavo, G., 2000. C-terminal half of tetanus toxin fragment C is sufficient for neuronal binding and interaction with a putative protein receptor. Biochem J 347 Pt 1, 199-204.

Khushoo, A., Pal, Y., Singh, B.N., Mukherjee, K.J., 2004. Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II. Protein Expr Purif 38, 29-36.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Maassen, C.B.M., Laman, J.D., Heijne den Bak-Glashouwer, M.J., Tielen, F.J., van Holten-Neelen, J.C.P.A., Hoogteijling, L., et al., 1999. Instruments for oral disease-intervention strategies: recombinant Lactobacillus casei expressing tetanus toxin fragment C for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis. Vaccine 17, 2117-2128.

Makoff, A.J., Oxer, M.D., Romanos, M.A., Fairweather, N.F., Ballantine, S., 1989. Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons. Nucleic Acids Res 17, 10191-10202.

Miyake, R., Kawamoto, J., Wei, Y.L., Kitagawa, M., Kato, I., Kurihara, T., et al., 2007. Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. Appl Environ Microbiol 73, 4849-4856.

Morris, N.P., Consiglio, E., Kohn, L.D., Habig, W.H., Hardegree, M.C., Helting, T.B., 1980. Interaction of fragments B and C of tetanus toxin with neural and thyroid membranes and with gangliosides. J Biol Chem 255, 6071-6076.

Ribas, A.V., Ho, P.L., Tanizaki, M.M., Raw, I., Nascimento, A.L., 2000. High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli. Biotechnol Appl Biochem 31 ( Pt 2), 91-94.

Romanos, M.A., Makoff, A.J., Fairweather, N.F., Beesley, K.M., Slater, D.E., Rayment, F.B., et al., 1991. Expression of tetanus toxin fragment C in yeast: gene synthesis is required to eliminate fortuitous polyadenylation sites in AT-rich DNA. Nucleic Acids Res 19, 1461-1467.

Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

Schiavo, G., Ferrari, G., Rossetto, O., Montecucco, C., 1991. Tetanus toxin receptor specific cross-linking of tetanus toxin to a protein of NGF-differentiated PC 12 cells. FEBS Letters 290, 227-230.

Stevenson, A., Roberts, M., 2004. Intranasal immunisation against tetanus with an attenuated Bordetella bronchiseptica vector expressing FrgC: improved immunogenicity using a Bvg-regulated promoter to express FrgC. Vaccine 22, 4300-4305.

Studier, F.W., 1991. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of Molecular Biology 219, 37-44.

Tregoning, J.S., Nixon, P., Kuroda, H., Svab, Z., Clare, S., Bowe, F., et al., 2003. Expression of tetanus toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res 31, 1174-1179.

Vasina, J.A., Baneyx, F., 1996. Recombinant protein expression at low temperatures under the transcriptional control of the major Escherichia coli cold shock promoter cspA. Appl Environ Microbiol 62, 1444-1447.

Wang, M.Y., Zhang, Y.N., Lei, M., Zuo, D.M., Zhang, L.Y., Chen, Z.L., 2008. [Gene cloning, optimized expression and immunogenicity evaluation of tetanus toxin fragment C].
Nan Fang Yi Ke Da Xue Xue Bao 28, 731-735.

Yousefi, M., Khosravi-Eghbal, R., Hemmati, A., Shokri, F., 2013. Production and characterization of recombinant light chain and carboxyterminal heavy chain fragments of tetanus toxin. Avicenna J Med Biotechnol 5, 220-226.