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High-level Expression of Tetanus Toxin Fragment C in Escherichia coli | ||
| Archives of Razi Institute | ||
| مقاله 4، دوره 73، شماره 1، خرداد 2018، صفحه 27-38 اصل مقاله (506.71 K) | ||
| نوع مقاله: Original Articles | ||
| شناسه دیجیتال (DOI): 10.22092/ari.2018.114057 | ||
| نویسندگان | ||
| K. Aghaiypour* ؛ R. Teymourpour | ||
| Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute, Agricultural Research, Education, and Extension Organization, Karaj, Iran | ||
| چکیده | ||
| 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. | ||
| کلیدواژه‌ها | ||
| Clostridium tetani؛ Fragment C؛ pET22b expression vector؛ pET28a expression vector؛ E. coli BL21 (DE3) pLysS | ||
| عنوان مقاله [English] | ||
| بیان بسیار بالای قطعه C سم تتانوس در اشرشیا کلی | ||
| نویسندگان [English] | ||
| خسرو آقائی پور؛ رقیه تیمورپور | ||
| بخش ژنومیکس و مهندسی ژنتیک، مدیریت بیوتکنولوژی و آزمایشگاه مرکزی، موسسه تحقیقات واکسن و سرم سازی رازی، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران | ||
| چکیده [English] | ||
| قطعه C، انتهای کربوکسیل زنجیره سنگین سم تتانوس میباشد که میتواند پاسخ ایمنی علیه دوز کشنده این سم را تحریک نماید. این پلی پپتید میتواند به عنوان کاندیدای واکسن علیه عفونت تتانوس که توسط باکتری کلستریدیوم تتانی ایجاد میشود مطرح شود. این مطالعه بر روی مقایسه بیان قطعه C سم تتانوس در سلول BL21(DE3)pLysS که تحمل بالایی به سم تتانوس دارد با دو وکتور pET22b و pET28a به کمک آنالیزسدیم دودسیل سولفات پلی آکریل آمید ژل و وسترن بلات انجام شد. پس از استخراج DNA سویه هاروارد CN49205 کلستریدیوم تتانی، ژن هدف بکمک واکنش زنجیری پلیمراز تکثیر و سپس تعیین توالی شده و درون وکتورهای pET22b و pET28a کلون شد. سپس وکتورها داخل سلول BL21(DE3)pLysS تانسفورم شد و سپس توسط یک پروتکل از پیش بهینه شده بیان شد. سلولها توسط ایزوپروپیل بتا دی-1- تیو گالاکتوپیرانوزید (IPTG) در چهار تیمار 25، 30، 33 و 37 درجه سانتیگراد و سه زمان متفاوت 1، 2 و 3 ساعت بیان شد. اگرچهSDS-PAGE و وسترن بلات بیان قطعه C نوترکیب (r-fragment C) وارد شده به هر دو وکتور را تایید کرد اما وکتور pET28a نشان دهنده بیان بسیار بالاتر ( 66/38 میلی گرم بر لیتر در مفایسه با 33/32 میلی گرم بر لیتر pET22b بود (< 0.05 P). شرایط بیان بهینه پس از 3 ساعت تیمار با mM IPTG در 25 درجه سانتیگراد بود. نتایج نشان داد که وکتور pET28a و سلول BL21(DE3)pLysS تواناتر و سازگارتر برای بیان قطعه C سم تتانوس در مقایسه با وکتور pET22b و BL21(DE3)pLysS بود. این نتایج نگرشهای جدیدی برای بیان پروتئینهای سمی چون فرگمنت C به کمک فناوری نوترکیب در اشرشیا کلی را ارائه مینماید. | ||
| کلیدواژه‌ها [English] | ||
| کلستریدیوم تتانی, قطعه C, وکتور بیانی pET22b, وکتور بیانی pET28a, اشریشیا کلیBL21 (DE3) pLysS | ||
| مراجع | ||
|
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]. 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. | ||
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