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Purification of recombinant Mf cDPGS expressed in Escherichia coli . The codon-optimized cdpgs gene was cloned into the pET15b vector without an affinity tag, and expression was optimized in Escherichia coli BL21(DE3)-Codon-Plus. The recombinant protein was then purified by heat precipitation and size exclusion chromatography (SEC). Protein samples (5–10 μg) from each purification steps were analyzed by SDS-PAGE (12.5%) and the gel was stained with Coomassie Brilliant Blue. CE: Crude extract, HP 75 : Soluble fraction after heat precipitation (75°C, 30 min), SEC: Fraction after size exclusion chromatography. M: Protein marker, unstained protein ladder (Thermo Fisher Scientific, Carlsbad, USA).

Journal: Frontiers in Microbiology

Article Title: Establishment of an efficient one-step enzymatic synthesis of cyclic-2,3-diphosphoglycerate

doi: 10.3389/fmicb.2025.1601972

Figure Lengend Snippet: Purification of recombinant Mf cDPGS expressed in Escherichia coli . The codon-optimized cdpgs gene was cloned into the pET15b vector without an affinity tag, and expression was optimized in Escherichia coli BL21(DE3)-Codon-Plus. The recombinant protein was then purified by heat precipitation and size exclusion chromatography (SEC). Protein samples (5–10 μg) from each purification steps were analyzed by SDS-PAGE (12.5%) and the gel was stained with Coomassie Brilliant Blue. CE: Crude extract, HP 75 : Soluble fraction after heat precipitation (75°C, 30 min), SEC: Fraction after size exclusion chromatography. M: Protein marker, unstained protein ladder (Thermo Fisher Scientific, Carlsbad, USA).

Article Snippet: Competent cells of the E. coli expression strains – BL21 (DE3) and BL21 (DE3) CodonPlus-pRIL (Cam R , Merck, Darmstadt, Germany), and Rosetta (DE3) (Cam R , Stratagene, San Diego, USA) – were transformed with the constructed plasmids and grown in 200 mL – 400 mL lysogeny broth (LB, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) containing the appropriate antibiotics at different expression temperatures (37°C, 30°C, and 16°C).

Techniques: Purification, Recombinant, Clone Assay, Plasmid Preparation, Expressing, Size-exclusion Chromatography, SDS Page, Staining, Marker