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

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.

Establishment of a standard seed lot system of an Iranian Mumps virus strain; RS-12, for mass production of mumps and MMR vaccines.

BACKGROUND: At present the mumps virus strain used for production of mumps vaccine for our local use is Hoshino strain. However, according to our National public health policies, this strain should be replaced with a safer strain. Based on our previous data, the Iranian mumps strain; RS-12 has been proved to be the most suitable alternative to Hoshino strain with little or no adverse events following vaccination METHODS: The aim of the present study was to optimize propagation of RS-12 strain and prepare standard seeds for vaccine mass production. The virus was inoculated to cells using different methods and different multiplicity of infection (MOI). The viral suspensions were harvested using different methods. Quality control tests were run at different stages. RESULTS: Maximum viral yield was achieved when cell suspensions were inoculated at MOI of 1:10 and incubated at 36-37ºC for 48 hours, followed by replacement of the media and incubation at 33-34 ºC for 5-7 days. Filtration did not affect the viral titre. A standard seed lot system was successfully established and experimental batches of MMR vaccines were produced. CONCLUSION: The established seed lot system has met all requirements of WHO regulations and could be used in mass production of safe and efficacious mumps and MMR vaccines. Clinical trials are in progress for this newly produced vaccine.

Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase.

Bacterial L-asparaginases, enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Other substrates of asparaginases include L-glutamine, D-asparagine, and succinic acid monoamide. In this report, we present high-resolution crystal structures of the complexes of Erwinia chrysanthemi L-asparaginase (ErA) with the products of such reactions that also can serve as substrates, namely L-glutamic acid (L-Glu), D-aspartic acid (D-Asp), and succinic acid (Suc). Comparison of the four independent active sites within each complex indicates unique and specific binding of the ligand molecules; the mode of binding is also similar between complexes. The lack of the alpha-NH3(+) group in Suc, compared to L-Asp, does not affect the binding mode. The side chain of L-Glu, larger than that of L-Asp, causes several structural distortions in the ErA active side. The active site flexible loop (residues 15-33) does not exhibit stable conformation, resulting in suboptimal orientation of the nucleophile, Thr15. Additionally, the delta-COO(-) plane of L-Glu is approximately perpendicular to the plane of gamma-COO(-) in L-Asp bound to the asparaginase active site. Binding of D-Asp to the ErA active site is very distinctive compared to the other ligands, suggesting that the low activity of ErA against D-Asp could be mainly attributed to the low k(cat) value. A comparison of the amino acid sequence and the crystal structure of ErA with those of other bacterial L-asparaginases shows that the presence of two active-site residues, Glu63(ErA) and Ser254(ErA), may correlate with significant glutaminase activity, while their substitution by Gln and Asn, respectively, may lead to minimal L-glutaminase activity.

Do bacterial L-asparaginases utilize a catalytic triad Thr-Tyr-Glu?

The structures of Erwinia chrysanthemi L-asparaginase (ErA) complexed with the L- and D-stereoisomers of the suicide inhibitor, 6-diazo-5-oxy-norleucine, have been solved using X-ray crystallography and refined with data extending to 1.7 A. The distances between the Calpha atoms of the inhibitor molecules and the hydroxyl oxygen atoms of Thr-15 and Tyr-29 (1.20 and 1.60 A, respectively) clearly indicate the presence of covalent bonds between these moieties, confirming the nucleophilic role of Thr-15 during the first stage of enzymatic reactions and also indicating direct involvement of Tyr-29. The factors responsible for activating Tyr-29 remain unclear, although some structural changes around Ser-254', Asp-96, and Glu-63, common to both complexes, suggest that those residues play a function. The role of Glu-289' as the activator of Tyr-29, previously postulated for the closely related Pseudomonas 7A L-glutaminase-asparaginase, is not confirmed in this study, due to the lack of interactions between these residues in these complexes and in holoenzymes. The results reported here are consistent with previous reports that mutants of Escherichia coli L-asparaginase lacking Glu-289 remain catalytically active and prove the catalytic roles of both Thr-15 and Tyr-29, while still leaving open the question of the exact mechanism resulting in the unusual chemical properties of these residues.