S c easycomp transformation kit

S. cerevisiae strain Y258 from Open Biosystems was used with the S.c. EasyComp Transformation Kit (Invitrogen) for generating chemically competent yeast, and the expression vectors were transformed into these yeasts following the manufacturer’s protocol. Yeasts carrying the expression vectors were cultured at 30 °C in synthetic -Ura dropout medium (0.67% yeast nitrogen base with ammonium sulfate, 0.077% -Ura dropout supplement, and 2% raffinose). Once absorbance reached 1.0 to 1.2 at 600 nm, 50% of the culture volume of 3× expression medium (3% yeast extract, 6% tryptone, and 6% galactose) was added, and incubation was continued for 6 h before harvesting cells by centrifugation at 5000g and 4 °C for 10 min.

A bacterial culture E. coli BL21 (DE3) pLysS (Novagen, Darmstadt, Germany) containing GST-FaCCR or GST (control) was grown in LB with appropriate antibiotics at 37°C, with shaking at 150 rpm. When OD₆₀₀ reached 0.5–0.6, IPTG was added to a final concentration of 0.2 mM to the bacterial culture at 16°C, with shaking at 150 rpm for 16–18 h. GST-FaCCR was purified using the GST Bind resin (Novagen, Darmstadt, Germany) according to the manufacturer's instructions. In addition, the resulting plasmid GST-POD was transformed into E. coli Rosetta (DE3)pLysS cells (Novagen, Darmstadt, Germany). The recombinant protein was expressed and isolated as described (Ring et al., 2013 (link)). Besides, pYES2-FaCAD was transformed into yeast cells (S. cerevisae INVSc.1) using the S. c. EasyComp Transformation Kit (Invitrogen, Darmstadt, Germany). Positive clones were confirmed by PCR screen, and inoculated in a SC-U selective medium plus 2% galactose to express pYES2-FaCAD in yeast cells. Protein extraction was carried out according to the manufacturer's instructions. The supernatant was used for enzymatic assays. Expression and purification of FaPOD27 was performed as described (Ring et al., 2013 (link)).

Proofreading cDNA amplicons were ligated into the pYES2.1/V5-His-TOPO vector (Invitrogen, Paisley, UK). Sense constructs were isolated and confirmed by sequencing. The vectors harbouring the DFR cDNAs Ang.DFR2 and Ang.DFR1 were transformed into yeast strain INVSc1 using the Sc. EasyComp Transformation Kit (Invitrogen, Carlsbad, CA). Preparation of the protein fractions was performed using a modified protocol according to Pompon et al. [59] (link). Protein fractions were shock frozen in liquid nitrogen and stored at −80°C.

The yeast display vector was generated as previously described by Van Blarcom et al.^{23 (link)}. A negative control plasmid containing no gene of interest but expressing the V5 tag was also generated. Briefly, proteins of interest were displayed on S. cerevisiae strain BJ5465 by transformation of the yeast display vector containing the protein coding sequence using S.C. EasyComp™ Transformation Kit (Invitrogen Cat# K505001) and selected on CM Glucose minus Tryptophan agar plates (Teknova Inc. C3060) for 4 days at 30 °C. Individual colonies were confirmed to contain the vector by PCR using gene specific primers and cultured in SDCAA media (Teknova Inc. 2S0540) overnight at 30 °C, 250 rpm and then shifted to SGCAA (Teknova Inc. 2S0562) media to induce protein expression for 18–24 h at 20 °C, 250 rpm. Cells were harvested, washed with ice-cold phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (PBSB) and resuspended to a final concentration of 3 × 10⁷ cells/ml.

Preparation and transformation of competent S. cerevisiae cells was performed using S. c. EasyComp™ Transformation Kit (Invitrogen) according to manufacturer’s protocol. Transformant selection was performed on plates containing minimal medium without uracil (SC-U) with 2% glucose and G418 antibiotic for 3 days at 28 °C. To induce protein expression, the transformants were grown in SC-U medium with 2% galactose and G418. After 2–4 days of growth the yeast cells were collected for polyisoprenoid and CPY analyses. In vivo complementation assay of rer2Δ mutation with AtCPT1 was performed as described previously [24 (link)].

For heterologous expression of apo-aequorin, yeast strains were transformed with the multicopy URA3-based plasmid pYX212-cytAEQ harboring the apoaequorin cDNA under the control of the strong TPI yeast promoter [54 (link)]. Plasmid pYX212-cytAEQ was a generous gift from E. Martegani and R. Tisi (University of Milano-Bicocca, Milan, Italy). Yeast transformation [72 (link)] was performed using S.c. EasyComp™ Transformation Kit (Invitrogen, Catalog number: K505001) following the manufacturer’s indications.

Primers containing HindIII (forward) and XhoI (reverse) restriction sites (underlined in Table S1) OVD9VF and OVD9VR (Scd) were used to amplify the ORF of the O. vulgaris Scd, using the high fidelity Pfu Turbo DNA polymerase (Promega). Further cloning into the yeast expression vector pYES2 (Invitrogen), which contains a GAL1 promoter that is inducible by galactose and has URA3 as selective marker, was achieved after ligation of restricted ORF amplicons and plasmid pYES2 to produce the construct pYES2-Scd. The recombinant plasmids pYES2-Scd or pYES2 empty (negative control) were transformed into S. cerevisiae competent cells InvSc1 (S.c. EasyComp Transformation Kit, Invitrogen). Yeast were grown in SC-ura for 3 days.
One single yeast colony containing the pYES2-Scd or pYES2 was grown overnight at 30 °C in 5 mL of liquid SC-ura. Cell cultures were then used to inoculate 10 mL of fresh SC-ura for a final OD₆₀₀ of 0.4. Four replicates for each construct (pYES2-Scd or pYES2) were run. Cells were grown at 30 °C for 5 h before the expression of the transgene was induced by the addition of galactose to 2% (w/v) [46 (link)]. After 48 h of galactose induction, yeast samples were collected, washed and homogenised in chloroform/methanol (2:1, v/v) containing 0.01% BHT. Samples were kept at −20 °C until further analysis.