bacmid dna  (Expression Systems Inc)

Journal: Plant Direct

Article Title: The Structure of the Chemotype Determining Locus in Cannabis sativa

doi: 10.1002/pld3.70166

Figure Lengend Snippet: Functional characterization of recombinant cannabinoid oxidocyclases. Purified proteins, secreted from sf9 cells infected with recombinant baculovirus, were functionally characterized. All assays were supplemented with CBGA precursor. Activity presented as percent of total CBGA converted was normalized against canonical CBCAS activity at 100% functionality. With CBCA as the dominant product of these enzymes, comparative analysis of activity for Clade C.4, Clade C.5, and Clade C.6 was carried out against CBCAS. In assays with purified protein from sf9 expression lower activity of CBCA production was observed in Clade C recombinant enzymes. Clade C.4 comparative activity was 26% of CBCAS, while Clade C.5 and Clade C.6 were 16% and 11%, respectively, of CBCAS.

Article Snippet: Purified bacmid DNA (~1 μg/μL) was transfected into Sf9 cells (8 × 10 5 cells/mL) using ExpiFectamine Sf Reagent following recommendations from the Bac‐to‐Bac expression system manual (Gibco).

Techniques: Functional Assay, Recombinant, Purification, Infection, Activity Assay, Expressing

Journal: Plant Direct

Article Title: The Structure of the Chemotype Determining Locus in Cannabis sativa

doi: 10.1002/pld3.70166

Figure Lengend Snippet: Full chromatograms for cannabinoid oxidocyclase activity from sf9 expressed protein in vitro assays. HPLC‐UV graphical chromatograms from in vitro enzymatic assays showcasing the activity of sf9 secreted recombinant cannabinoid oxidocyclases, plotting the absorbance against retention time for samples (A–F). (A) 17‐Cannabinoid mix from certified reference standard: This chromatogram serves as a reference, displaying peaks corresponding to various cannabinoids with their respective retention times. Peaks are labeled with numbers (1–17) for available neutral and acidic cannabinoid standards. (B) CladeC.4: a single major peak at Peak 7, corresponding to CBGA, and a minor peak at Peak 17, corresponding to CBCA, indicating the production of a small amount of CBCA by the enzyme. (C) CladeC.5: prominent CBGA peak at Peak 7 and a minor CBCA peak at Peak 17, although with a slightly lower abundance compared to CladeC.4. (D) CladeC.6: a dominant CBGA peak at Peak 7, with a minor abundance of CBCA at Peak 17, similar to CladeC.4 and CladeC.5. (E) CBCAS: In contrast to the previous samples, while a dominant peak of CBGA is present at Peak 7, an increased CBCA production is observed for CBCA at Peak 17, indicating significant production of CBCA by CBCAS relative to tested clade C genes. (F) No enzyme control: This control reaction contained no enzyme, and only the CBGA peak at Peak 7 was observed.

Article Snippet: Purified bacmid DNA (~1 μg/μL) was transfected into Sf9 cells (8 × 10 5 cells/mL) using ExpiFectamine Sf Reagent following recommendations from the Bac‐to‐Bac expression system manual (Gibco).

Techniques: Activity Assay, In Vitro, Recombinant, Labeling, Control