Materials and reagents
E. coli Top10 and E. coli BL21 (DE3) were purchased from Novagen, Inc. (San Diego, CA, USA) and stored in our lab. The expression vector pCold-TF was from Takara Bio (Dalian, China). Restriction enzymes NdeI and SalI were from Thermo Fisher (MA, USA). Ni–NTA Agarose was from Thermo Fisher (Invitrogen, USA). Thrombin CleanCleave™ Kit was purchased from Sigma Aldrich (St Louis, MO, USA). Micro Protein PAGE Recovery Kit was from Sangon Biotech. Co. Ltd. (Shanghai, China). Other chemicals were of analytical or higher grade available.
Cloning of Rab3A gene from Rattus norvegicus
Rattus norvegicus hippocampal tissues were used as Rab3A gene source. The total RNA was extracted from the hippocampal tissues and the first-strand DNA synthesis was performed according to the instructions of cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). The DNA was used as the template to obtain Rab3A gene by PCR amplification with the forward primer containing NdeI recognition site (5ʹ-GGGAATTCCATATGGCCTCAGCCACAGACTCTC-3ʹ) and the reverse primer containing SalI recognition site (5ʹ-ACGCGTCGACTCAGCAGGC GCAATCCTGAT-3ʹ). PCR was completed under the following conditions: preincubation for 3 min at 98 °C, followed by 32 cycles of 20 s at 98 °C, 20 s at 60 °C, 40 s at 68 °C, and then a final extension step of 72 °C for 30 min. The PCR products were subjected to electrophoresis on a 1.2 % agarose gel. The recovered and purified PCR fragment with a size of about 660 bp was ligated into pMD18T vector and then transformed into E. coli Top10, which were incubated in 1 ml LB fluid medium at 37 °C for 45 min with shaking (220 rpm) and then plated onto LB agar plates containing ampicillin (100 µg/ml). The single positive colonies were picked out and the plasmids were extracted. After digestion with NdeI and SalI, the positive colonies were confirmed by DNA sequencing. Recombinant plasmid (pMD18T-Rab3A) was digested with restriction endonucleases NdeI and SalI and the resulting Rab3A gene fragments were ligated into the expression vector pCold-TF which had been digested with the same enzymes. Then the ligated plasmid pCold-TF-Rab3A was transformed into E. coli BL21(DE3).
Expression and purification of Rab3A fusion protein
The recombinant plasmids encoding Rab3A were transformed into E. coli BL21 (DE3) by heat shock and the cells were plated onto LB agar plates with 100 µg/ml ampicillin, followed by incubation overnight at 37 °C. Single colonies from plates were transferred to 5 ml LB fluid medium containing 100 µg/ml ampicillin and incubated overnight at 37 °C with shaking at 220 rpm. This culture was diluted 1:100 into 500 ml LB broth plus ampicillin (100 µg/ml). The cells were grown at 37 °C until an OD600 of 0.6–1.0 was reached. After the temperature was reduced to 16 °C, the recombinant expression was induced by the addition of IPTG at a final concentration of 0.5 mM and the cells were grown overnight in an incubator with constant shaking of 220 rpm. The cells were harvested by centrifugation at 4000 g for 20 min at 4 °C. Cell pellet was re-suspended in a lysis buffer at a ratio of 5 ml buffer per 1 g cell pellet, followed by sonication. After centrifugation at 4000g at 4 °C, the supernatant was transferred into 15 ml Falcon tubes containing 200 µl of Ni–NTA Agarose and incubated on a rotating wheel for 3 h at 4 °C. The Ni–NTA Agarose with bound protein was separated from the lysate by centrifugation at 500g at 4 °C and washed three times. In the final step, the bound proteins were eluted from the Ni–NTA Agarose with 400 µl of 50 mM Hepes (pH 8.0) buffer containing 500 mM NaCl and 250 mM imidazole (elution buffer). An aliquot of the eluted proteins were analyzed by SDS-PAGE.
SDS-PAGE and western blot analysis
Samples of Rab3A heterologous expression and purification were resolved on a 10 % SDS-PAGE gel in principle as described by Laemmli (1970), followed by visualization with Coomassie brilliant blue staining and scanning with a G:BOX Gel imaging system (Syngene, Cambridge, UK). For further identification of the expressed Rab3A fusion protein, the protein in the corresponding band was transferred from gel lane onto a nitrocellulose membrane (PALL Corporation, USA) using a blot electrotransfer apparatus in the wet transfer method (100 mA/2.5 h) and blocked in 5 % milk/TBST (50 mM Tris–HCl, 150 mM NaCl, 0.1 % Tween-20, pH 7.5) for 1.5 h at room temperature, and then probed with the mouse anti-His tag antibody (Novex, Life Technology, USA) (1:5000 dilution in 5 % milk/TBST) for 1.5 h at room temperature. After the membrane was washed three times (each for 6 min) using TBST, it was incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (Promega Corporation, Madison, WI, USA) (1:8000 dilution in 5 % milk/TBST), followed by washing with TBST extensively. The blot was developed using the enhanced chemiluminescence (ECL) method (Thermo Scientific, USA) and recorded with a ChemiDoc XRS imaging system (Bio-Rad, USA).
Antibody generation of Rab3A fusion protein and western blot analysis
The commonly used immunization protocols were employed to generate polyclonal antibody to Rab3A protein (Cooper and Paterson 2001). Briefly, the recombinant Rab3A (about 800 µg/ml in PBS) was mixed with equal volume of Freund’s incomplete adjuvant and injected subcutaneously into two 4- to 6-month-old mice. Initial immunization was followed by three booster injections at 2-week intervals. Antiserum was collected from the animals after 14 weeks. Antibody verification using western blotting was performed similarly to the identification of the expressed Rab3A fusion protein described as above, with the newly generated polyclonal antibody as the first antibody instead of anti-His tag antibody (1:4000 dilution in 5 % milk/TBST). Both recombinant and native Rab3A proteins were used as antigens.
Tag sequence removal and de-tagged Rab3A protein purification
In order to remove the tag sequence from Rab3A fusion protein, the purified fusion protein was cleaved with thrombin. For optimizing the experimental conditions, the enzymolysis was performed and compared at different thrombin concentrations and for different times. After enzymolysis, the resulting protein mixture were electrophoresed on a 12 % SDS-PAGE, followed by gel protein recovery using a Micro Protein PAGE Recovery Kit according to the manufacturer’s instructions.
GST pull-down and western blotting
GST pull-down experiment was used to detect a representative Rab3A-mediated protein interaction. For this detection, GST-Syt I-C2AB (the cytoplasmic C2 domains of synaptotagmin I) preparation and GST pull-down assay were performed according to the procedures previously described (Chapman and Jahn 1994). Glutathione-Sepharose bead-bound Syt I-C2AB was incubated with recombinant Rab3A protein overnight at 4 °C in a HNa buffer (10 mM Hepes–NaOH, 150 mM NaCl, 1 µM pepstatin A, 2 µM leupeptin, 0.3 mM phenylmethylsulfonyl fluoride, pH 7.4) containing 0.5 % Triton X-100. After incubation, the Glutathione-Sepharose beads were washed three times each with 1 ml of the HNa buffer containing 0.1 % TritonX-100. The proteins that remained bound to the beads were eluted with 2× SDS loading buffer and were analyzed by 10 % SDS-PAGE, followed by immunoblotting with the anti-Rab3A polyclonal antibody we prepared and anti-GST antibody (Abcam, England) as the primary antibodies, and horseradish peroxidase (HRP)-conjugated anti-mouse lgG antibody as the second antibody.