Abstract
BackgroundRab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis. Up to date, how Rab3A participates in such a regulatory process is not completely clear.ResultsIn this report the Rab3A gene from Rattus norvegicus was cloned and heterologously expressed in E. coli using pCold-TF expression vector with folding capacity. Due to the presence of His-tag sequence on the N-terminal side, Rab3A fusion protein was purified to greater than 95 % purity with a single Ni-affinity purification step. After the Rab3A fusion protein was used to immunize mice, an anti-serum against Rab3A with a titer of about 6000 was generated. Western blot analysis indicated that the prepared polyclonal antibody could recognize both Rab3A fusion protein and native Rab3A protein. To remove the tag sequence, thrombin was used to cleave the Rab3A fusion protein, followed by SDS-PAGE to separate the cleavage products. Using the gel protein recovery strategy with a Micro Protein PAGE Recovery Kit, the de-tagged Rab3A protein of electrophoretic purity was prepared.ConclusionsThe present work not only prepared the ground for the study on Rab3A-mediated protein interactions, but also provided systematic experimental methods referable for the similar studies.
Highlights
Rab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis
The gene was successfully cloned into the expression vector pCold-trigger factor (TF) and transformed into E. coli BL21(DE3) for heterologous expression
We used a fusion expression vector newly developed by Takara Bio Inc. (Shiga, Japan). pCold TF is a fusion cold shock expression vector that expresses trigger factor chaperone as a soluble fusion tag
Summary
Rab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis. The major Rab protein in brain is Rab3A, which is able to associate with synaptic vesicle membranes by a C-terminal lipid modification (Geppert et al 1994; Johnston et al 1991). This protein associates with synaptic vesicles in its GTPbound form and dissociates from the vesicles upon GTP hydrolysis or depolarization of the nerve terminal (Fischer von Mollard et al 1994). How Rab3A participates in the regulation of synaptic vesicle exocytosis is not completely clear It most likely acts via its effector proteins.
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