DOI: 10.6084/m9.figshare.22199413
title: C24-SMT assay (Michellod et al. 2023)

description: <strong>Heterologous gene expression. </strong>To determine the activity of the putative C24-SMT enzymes, we overexpressed OalgSMT, OclaSMT and ArathSMT1 in <em>E. coli</em> and performed enzymatic assays. GenScript (Genscript®) generated pet28a(+) (NheI/XhoI) plasmid containing the sequence of interest. For expression, the pet28(a)-OalgSMT and pet28(a)-OclaSMT vectors were transformed in Lemo21(DE3) <em>E. coli</em> competent cells (New England Biolabs (NEB)). The pet28(a)-ArathSMT1 vector was transformed in Overexpress C43(DE3) pLysS <em>E. coli</em> competent cells (Lucigen). A single colony of transformed cells was grown in 3 mL lysogeny broth (LB) supplemented with the appropriate antibiotics for 8 h (37°C, 150 rpm). 1 mL pre-culture was used to inoculate 1 L ZYP-5052-Rich-Autoinduction-Medium (<em>27</em>) supplemented with antibiotics and 1500 µM rhamnose. The cultures were grown for 72 h at 20°C, with rotation at 150 rpm. Cells were harvested by centrifugation at 4,500 × g for 25 min at 4°C, the supernatant discarded and the resulting pellet stored at -20°C until further use. <strong>Protein extraction/cell lysis. </strong>The frozen pellets were thawed on ice. They were then resuspended in 15 mL sucrose solution (750 mM sucrose solution in 20 mM phosphate buffer at pH 7.5). Once the pellets were dissolved in the sucrose solution, 5 mg lysozyme was added and the tubes shaken for 10 min at RT. Cells were lysed by addition of 30 mL lysis buffer (100 mM NaCl, 15% glycerol, 3 mM EDTA in 20 mM phosphate buffer at pH 7.5), 0.2 mL MgSO4 stock solution (120 g L-1) and 0.3 mL Triton X-100. The mix was vigorously shaken and incubated on ice until it reached a gelatinous consistency. The DNA was fragmented by addition of 2 mL DNase stock solution (50 mg DNase I in 35 mL buffer (20 mM Tris-HCl pH8, 0.5 M NaCl) and 15 mL glycerol). Finally, the cell fragments and inclusion bodies were pelleted by centrifugation (45 min, 16,000 × g, 4°C). The supernatant, containing soluble proteins, was aliquoted and stored at -80°C until further use. The overexpression yielded a target protein migrating on SDS-PAGE with the expected size of ~40 kDa. <strong>Enzymatic assay. </strong>We tested ten sterol substrates (cycloartenol, lanosterol, zymosterol, lathosterol, 7-dehydrocholesterol, desmosterol, campesterol, cholesterol, sitosterol and 24-methylene-cholesterol). The assay was performed in 600 µL total volume. 100 µL crude soluble protein extract was mixed with 400 µL phosphate buffer (20 mM, 5% glycerol, pH 7.5) in a 15 mL tube containing a sterol substrate (final concentration 100 µM) dispersed in Tween 80 (0.1% v/v). The reaction was initiated with 100 µL SAM working solution (0.6 mM). The reaction was performed in a water bath at 35°C for 16 h. The reaction was terminated with 600 µL 10% methanolic KOH. The products were extracted three times with 2.5 mL hexane and mixed on a vortex for 30 s. The resulting organic layer was evaporated to dryness in a concentrator at 30°C, V-AL for 1.5 h. Two internal standards, 5α-cholestane (100 µL, 1 mM solution) and ribitol (40 µL 200 mg/L solution), were added to the tubes and evaporated to dryness. The samples were derivatized and analyzed on an Agilent GC-MS as described above or directly analyzed on a QExactive Plus Orbitrap (Thermo Fisher Scientific) equipped with a HESI probe and a Vanquish Horizon UHPLC system (Thermo Fisher Scientific). The sterols were separated on a C18 column (Accucore Vanquish C18+, 100 x 2.1, 1.5 µm, Thermo Fischer Scientific). For method details please refer to Michellod et al. 2023.