Gene cloning, expression and polyclonal antibody preparation of Rab3A for protein interaction analysis

Abstract

Background: Rab3A 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.

Results: In 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.

Conclusions: The 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.

Keywords: Fusion expression; Gel protein recovery; Polyclonal antibody; Protein interaction; Rab3A.

Fig. 1

Agarose gel electropherograms of the products of Rab3A PCR amplification (a) and the recombinant vector digestion by NdeI and SalI (b). Lanes labelled with 1 in (a) and (b) represent the products of PCR amplification and double digestion, respectively. M, DNA molecular weight marker

Fig. 2

Induction expression, affinity purification and confirmation of Rab3A fusion protein. Lane 1, total proteins (35 μg) before induction; lane 2, total proteins (35 μg) after induction with IPTG; lane 3, the purified Rab3A fusion protein; lane 4, western blotting of Rab3A fusion protein. M, protein molecular weight marker

Fig. 3

Titer determination of antiserum against Rab3A fusion protein. 1, negative control; 2–7, 1:1000, 1:2000,1:3000, 1:4000, 1:5000 and 1:6000, respectively

Fig. 4

Western blot detection of Rab3A fusion protein (a) and native Rab3A protein (b) with prepared polyclonal antibody. Lanes 1 and 2 in (a) were Rab3A fusion protein with different loading amounts

Fig. 5

SDS-PAGE of Rab3A fusion protein before and after cleavage with thrombin. Lanes 1 and 2, Rab3A fusion protein with different loading amounts before cleavage (10 μg per lane). Lane 3, protein mixture after cleavage with thrombin (10 μg per lane). Lanes 4 and 5, purified de-tagged Rab3A protein with different loading amounts

Fig. 6

Detection of Rab3A interaction with the C2 domains of synaptotagmin I