FIGURE 2.

Production of recombinant enzymes. A, Coomassie-stained SDS-PAGE of purified StrepII-CsaA-His₆ (left panel) and StrepII-CsaC-His₆ (right panel). B, to select the construct most suited for the production of active recombinant CsaB, the wild type and a codon-optimized (CsaB_co) version of the CsaB sequence were cloned (full-length or after N-terminal truncation, Δ69) to produce proteins with tags on both (StrepII and His₆) or only one end (His₆), as indicated. Transformed bacteria were lysed, separated into soluble (s) and insoluble (i) fractions, and fractions were separately run on PAGE. After transfer onto nitrocellulose, the blot was developed with an anti-penta-His antibody. m, marker. C, soluble fractions were used to measure CsaB activity in a radioactive incorporation assay. D, Coomassie-stained gel demonstrating the purification result for Δ69-CsaB_co-His₆ (IMAC, immobilized metal ion affinity chromatography; SEC, size exclusion chromatography).