CLONING, EXPRESSION AND PURIFICATION OF SNAP-25 PROTERIN

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MOUSAVI M.L.; NAZARIAN SH.; AMANI J.; SOROURI ZANJANI R.

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Journal: Journal of Advances in Medical and Biomedical Research Year:2007 | Volume:14 | Issue:58 Page(s): 1-10

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Title

CLONING, EXPRESSION AND PURIFICATION OF SNAP-25 PROTERIN

Author(s)

MOUSAVI M.L. | NAZARIAN SH. | AMANI J. | SOROURI ZANJANI R. | Issue Writer Certificate

Keywords

BOTULINUM NEUROTOXIN TYPES AQ3

AND EQ2

RECOMBINANT PROTEINQ3

SNAP-25Q3

Abstract

Background & Objectives: Clostridial neurotoxin inhibits neurotransmitter release by selective and specific intracellular proteolysis of synaptosomal associated protein of 25KDa (SNAP-25), synaptobrevin/VAMP-2 and syntaxin. SNAP-25 is one of the components that forms docking complex in synaptic ends. This protein is subtrate for botulinum neurotoxins types A, C, AND E. Each of these toxin serotypes specifically cleaves SNAP-25 in a particular position and thereby blocks docking and synaptic vesicle membrane fusion and finally prevents neurotransmitter exocytosis and transition of neurotic signals. Recombinant production of SNAP-25 in the laboratory can be used as a subtrate for the detection of clostridium botulinum types A, AND E neurotoxins.Materials & Methods: In order to use the protein as a subtrate for detection of different types of clostridium neurotoxins in-vitro the protein was produced by recombinant technique. The cDNA from SNAP-25 was synthesized from total RNA purified from frozen Rattus norvegicus brain and amplified by RT-PCR the amplified fragment was cloned into pET32a expression vector. The identity of RECOMBINANT PROTEIN was confirmed by Western blot using specific antibody and finally the RECOMBINANT PROTEIN was purified through an affinity column chromatography (Ni-NTA).Results: The optimum conditions of expression of SNAP-25 were found to be IPTG (1mM) and incubation at 37oc for 5 hours. The RECOMBINANT PROTEIN was isolated and purified using Ni-NTA column with imidazole at a concentration of 25OmM. Using enterokinase to cut the fision at 37oC comparatively yielded better results than room temperature.Conclusion: The protein retained its structure during the purification process being suitable for cutting and further tests. The purified protein we obtained can be used as substrate for detection of clostridium botulinum types A, AND E toxins.

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APA: Copy

MOUSAVI, M.L., NAZARIAN, SH., AMANI, J., & SOROURI ZANJANI, R.. (2007). CLONING, EXPRESSION AND PURIFICATION OF SNAP-25 PROTERIN. JOURNAL OF ADVANCES IN MEDICAL AND BIOMEDICAL RESEARCH, 14(58), 1-10. SID. https://sid.ir/paper/61559/en

Vancouver: Copy

MOUSAVI M.L., NAZARIAN SH., AMANI J., SOROURI ZANJANI R.. CLONING, EXPRESSION AND PURIFICATION OF SNAP-25 PROTERIN. JOURNAL OF ADVANCES IN MEDICAL AND BIOMEDICAL RESEARCH[Internet]. 2007;14(58):1-10. Available from: https://sid.ir/paper/61559/en

IEEE: Copy

M.L. MOUSAVI, SH. NAZARIAN, J. AMANI, and R. SOROURI ZANJANI, “CLONING, EXPRESSION AND PURIFICATION OF SNAP-25 PROTERIN,” JOURNAL OF ADVANCES IN MEDICAL AND BIOMEDICAL RESEARCH, vol. 14, no. 58, pp. 1–10, 2007, [Online]. Available: https://sid.ir/paper/61559/en

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