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

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Archives of Razi Institute
Volume 73, Issue 1
March 2018
Pages 27-38

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