The full-length human complementary DNAs of b0,+AT (accession number: NM_001126335.1) and rBAT ( accession number: NM_000341.4) were subcloned into pCAG with N-terminal FLAG tag and pCAG with N-terminal 10× His tag, respectively. A standard two-step polymerase chain reaction was used to generate the mutations.

For protein expression, human embryonic kidney 293F cells (Invitrogen) were cultured in SMM 293T-I medium (Sino Biological Inc.) at 37°C under 5% CO2 in a Multitron-Pro shaker (130 rpm, Infors). For transfection, 3 mg of polyethylenimines (Polysciences), 0.75 mg of the b0,+AT plasmid, and 0.75 mg of the rBAT plasmid were preincubated for 15 min with fresh medium in a final volume of 50 ml and was added into 1 liter of cell culture whose cell density was ~2.0 × 106/ml. The transfected cells were harvested 48 hours after transfection.

For purification of the b0,+AT-rBAT complex, the cells were collected by centrifugation at 3800g for 10 min and resuspended in a buffer containing 25 mM tris (pH 8.0), 150 mM NaCl, and three protease inhibitors, aprotinin (0.2 μM, AMRESCO), pepstatin (1 μM, AMRESCO), and leupeptin (10.1 μM, AMRESCO). The membrane fraction was solubilized at 4°C for 2 hours with 2% (w/v) n-dodecyl-β-d-maltoside (Anatrace). Cell debris was removed by centrifugation at 18,700g for 45 min, and the supernatant was loaded onto anti-FLAG M2 affinity resin (Sigma-Aldrich). After the resin was rinsed with the wash buffer containing 25 mM tris (pH 8.0), 150 mM NaCl, and 0.05% glyco diosgenin (GDN) (w/v) (Anatrace), the protein was eluted with wash buffer plus FLAG peptide (0.2 mg/ml). Then, the eluent was loaded onto nickel resin (Ni-NTA, Qiagen) and washed with wash buffer plus 10 mM imidazole. The protein complex was eluted from the nickel resin with wash buffer plus 300 mM imidazole, following by concentrating and subjecting to size exclusion chromatography (SEC; Superose 6 Increase 10/300 GL, GE Healthcare) in buffer containing 25 mM tris (pH 8.0), 150 mM NaCl, and 0.02% GDN. The peak fractions were collected and concentrated for EM analysis and in vitro liposome-based transport assay.

For α-glucosidase activity measurement, the transfected cells were resuspended in a buffer containing 25 mM Hepes (pH 7.0), 150 mM NaCl, and the three protease inhibitors mentioned above. The buffer used for affinity chromatography and gel filtration was almost the same as that mentioned above except tris (pH 8.0) was replaced by Hepes (pH 7.0).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.