The ASFF1 CDS was amplified from S. pennellii LA0716 trichome cDNA using ASFF_F and ASFF_R primers (table S3) and cloned into the pGEM backbone using the pGEM-T Easy cloning kit (Promega, Madison, WI). The ASFF1 CDS was subsequently reamplified with the (pEAQ-HT)-ASFF-His_F and (pEAQ-HT)-ASFF-His_R primers (table S3) to add adapters for Gibson Assembly. The resulting PCR product was transferred to pEAQ-HT vector (41) previously digested with NruI–HF and SmaI restriction enzymes (NEB, Ipswich, MA) using 2× Gibson Assembly master mix (NEB, Ipswich, MA) according to the manufacturer’s instructions to create an expression clone coding for the full-length protein with a C-terminal 6× His tag (ASFF1-HT-pEAQ). The completed vector was subsequently transformed into LBA4404 cells, as described above. For transient expression, an Agrobacterium tumefaciens LBA4404 strain carrying the ASFF1-HT-pEAQ construct was streaked onto LB agar containing rifampicin (50 μg/ml) and kanamycin (50 μg/ml) and incubated for 3 days at 28°C. Single colonies were used to inoculate 250-ml Erlenmeyer flasks containing 50 ml of YEP medium with rifampicin (50 μg/ml) and kanamycin (50 μg/ml); cultures were incubated at 28°C and shaken at 300 rpm overnight. Cultures were harvested by centrifugation at 800g at 20°C for 20 min. The supernatant was discarded, and the resulting loose pellet was resuspended in 50 ml of buffer A [10 mM 2-ethanesulfonic acid (MES; Sigma-Aldrich, St. Louis, MO) at pH 5.6 and 10 mM MgCl2]. This cell suspension was centrifuged at 800g at 20°C for 20 min, and the resulting pellet was resuspended to a final OD600 = 1.0 with buffer A. A 200 mM solution of acetosyringone (Sigma-Aldrich, St. Louis, MO) dissolved in DMSO was added to the suspension at a final concentration of 200 μM, and the suspension was incubated at room temperature with gentle rocking for 4 hours. This suspension was infiltrated into fully expanded leaves of 6-week-old N. benthamiana plants using a needleless 1-ml tuberculin syringe. Plants were grown under a 16-hour photoperiod (70 μmol m−2 s−1 PPFD) and at a constant 22°C set to 70% relative humidity. At 8 days after infiltration, 28 g of infiltrated leaves was harvested, deveined, and flash-frozen in liquid nitrogen. Tissue was powdered under liquid nitrogen with a mortar and pestle and added to 140 ml of ice-cold buffer B [25 mM 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (EPPS) at pH 8.0, 1.5 M NaCl, 1 mM EDTA with 2 mM dithiothreitol (DTT), 1 mM benzamidine, 0.1 mM phenylmethansesulfonyl fluoride, 10 μM trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane (E-64), and 5% (w/v) polyvinylpolypyrrolidone (PVPP); all reagents were obtained from Sigma-Aldrich (St. Louis, MO) except DTT, which was obtained from Roche Diagnostics (Risch-Rotkreuz, Switzerland)]. The mixture was stirred for 4 hours at 4°C, filtered through six layers of Miracloth, and centrifuged at 27,000g at 4°C for 30 min. The supernatant was decanted and passed through a 0.22-μm polyethersulfone filter (EMD Millipore, Billerica, MA) before being loaded onto a HisTrap HP 1-ml affinity column and eluted using a gradient of 10 to 500 mM imidazole in buffer B using an ÄKTA start FPLC module (GE Healthcare, Uppsala, Sweden). Fractions were analyzed by SDS–polyacrylamide gel electrophoresis, and the presence of ASFF1-HT was confirmed by immunoblot using the BMG–His-1 monoclonal antibody (Roche, Mannheim, Germany) to detect His-tagged proteins. Purified ASFF1-HT was subsequently transferred to 100 mM sodium acetate (pH 4.5) with 50% glycerol using a 10DG desalting column (Bio-Rad, Hercules, CA). Protein was quantified against a standard curve of BSA (Thermo Fisher Scientific, Waltham, MA) using a modified Bradford reagent (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions.

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



Q&A
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.