Figure 2.

An SDS–PAGE analysis of the purified HbpS. The E. coli BL21 (DE3) pLysS strain containing the plasmid pETHbpS was grown and induced as described in §2. The cytoplasmic fraction containing His₆-tag-HbpS (lane 2) was incubated with Ni-NTA agarose. Unbound proteins remained in the flowthrough (lane 3). His₆-tag-HbpS was eluted in the presence of 250 mM imidazole (lane 4). The His₆ tag was then cleaved using TEV-protease; after a second Ni-NTA affinity chromatography, HbpS was then further purified by anion-exchange chromatography through DEAE-Sepharose (lane 5). The molecular weight (in kDa) of each of the protein markers is indicated (lane 1). A total of 5 mg purified His₆-tag-free HbpS was obtained from 1 l of bacterial culture.