CLONING, EXPRESSION AND PURIFICATION OF THE GENE CODING TRANSLOCATING DOMAIN OF BOTULINUM NEUROTOXIN TYPE A

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MOSSAVI M.L.; AMANI J.; TAVALAEI M.; KAMALI M.

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Journal: Pathobiology Research Year:2005 | Volume:7 | Issue:2 Page(s): 81-92

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Title

CLONING, EXPRESSION AND PURIFICATION OF THE GENE CODING TRANSLOCATING DOMAIN OF BOTULINUM NEUROTOXIN TYPE A

Author(s)

MOSSAVI M.L. | AMANI J. | TAVALAEI M. | KAMALI M. | Issue Writer Certificate

Keywords

CLOSTRIDIUM BOTULINUM TYPE AQ4

TRANSLOCATING DOMAINQ4

RECOMBINANT PROTEINQ3

Abstract

Purpose: Botulinum neurotoxin type A structurally consists of a 50KD light chain and a 100 KD heavy chain linked by a disulfide bound. The protein can be further divided into three functional domain: a catalytic domain corresponding to the light chain, a TRANSLOCATING DOMAIN associated with the N-terminal half of the heavy chain and a binding domain at the C-terminal half. To study the efficiency of TRANSLOCATING DOMAIN of Botulinum toxin type A independently or in association with the binding domain in order to achieve a protective protein, this domain of the toxin was produced by the recombinant technique. This may also offer an appropriate way for studying the structure and mechanism of the action of this domain of the toxin.Materials and Methods: Bacteria was grown in anaerobic condition and the genomic DNA was extracted by the alkaline method. After analyzing the sequence of the gene, the primer were designed. The gene was amplified by the PCR . The PCR product was analyzed by the enzyme digestion, then cloned into three expression vectors namely pRSETA, pET28a and pET32a. The expressed protein was analyzed on the SDS-PAGE and confirmed by the Elisa and Western blotting and then purified by the affinity chromatography.Results and Discussion: In this investigation the maximum expression was obtained at 0.5 mM IPTG, OD6oo: O.6 and 15 hours of incubation at 30°C after the induction with the IPTG. Though the expression of AT rich genes in E.coli system are generally low, but we had a good level of expression. The purification of the RECOMBINANT PROTEIN was difficult at the first step of purification because of the weak binding of the His-tag on the N-terminal of the protein to the column, but a 90% purification was achieved by the modification of the technique.

Cites

IMMUNOLOGICAL CHARACTERIZATION OF LIGHT CHAIN RECOMBINANT PROTEIN OF CLOSTRIDIUM BOTULINUM TYPE A

References

DETECTION OF CLOSTRIDIUM BOTULINUMTYPE A BY PCR-ELISA

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APA: Copy

MOSSAVI, M.L., AMANI, J., TAVALAEI, M., & KAMALI, M.. (2005). CLONING, EXPRESSION AND PURIFICATION OF THE GENE CODING TRANSLOCATING DOMAIN OF BOTULINUM NEUROTOXIN TYPE A. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), 7(2), 81-92. SID. https://sid.ir/paper/81161/en

Vancouver: Copy

MOSSAVI M.L., AMANI J., TAVALAEI M., KAMALI M.. CLONING, EXPRESSION AND PURIFICATION OF THE GENE CODING TRANSLOCATING DOMAIN OF BOTULINUM NEUROTOXIN TYPE A. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES)[Internet]. 2005;7(2):81-92. Available from: https://sid.ir/paper/81161/en

IEEE: Copy

M.L. MOSSAVI, J. AMANI, M. TAVALAEI, and M. KAMALI, “CLONING, EXPRESSION AND PURIFICATION OF THE GENE CODING TRANSLOCATING DOMAIN OF BOTULINUM NEUROTOXIN TYPE A,” PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), vol. 7, no. 2, pp. 81–92, 2005, [Online]. Available: https://sid.ir/paper/81161/en

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