FIG 3.

Purification and enzymatic activity of E. coli extracts expressing L. plantarum vprA and recombinant VprA protein. (A) SDS-PAGE analysis of the expression and purification of the His₆-tagged VprA. Data represent results of analysis of soluble cell extracts of IPTG-induced E. coli BL21(DE3)(pURI3-Cter) (lane 1) or E. coli BL21(DE3)(pURI3-Cter-VprA) (lane 2), flowthrough from the affinity resin (lane 3), or fractions eluted after His affinity resin (lanes 4 to 8). The 10% gel was stained with Coomassie blue. Molecular mass markers are located on the left (SDS-PAGE standards; Bio-Rad). (B) HPLC chromatograms showing vinylphenol reductase activity of soluble cell extracts of IPTG-induced E. coli BL21(DE3)(pURI3-Cter) (control) or E. coli BL21(DE3)(pURI3-Cter-VprA) (VprA) incubated in 1.5 mM 4-vinylphenol and 15 mM NADH. HPLC chromatograms also showed the reductase activity of purified His₆-VprA protein (500 μg) (VprA) or the reaction mix without VprA protein (control). The 4-vinylphenol (VP) and 4-ethylphenol (EP) detected are indicated. Chromatograms were recorded at 280 nm.