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Fig. 3 | BMC Microbiology

Fig. 3

From: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase

Fig. 3

Purification and functional characterization of HepP. a Expression and purification of recombinant HepP (rHepP) in E. coli. Plasmid pND1 containing the hepP structural gene was expressed from the arabinose-inducible promoter pBAD. Induction experiments were conducted using 0.02% L-arabinose as described in Methods using the E. coli TOP10 strain as expression host. Proteins were purified by nickel column chromatography under native conditions, separated by 10% SDS PAGE and stained with Coomassie blue. Image is a compilation of lanes from the same gel. Lanes: M, molecular weight standard in kDa; L, whole cell lysate; W1 and W2, first and second of four column washes; E3 and E4, third and fourth of four elution fractions containing rHepP. b Immunoblotting to confirm rHepP purification. Equal amounts of purified proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-V5 antibody (Invitrogen). The probed membranes were treated with anti-mouse horseradish peroxidase-conjugated IgG and developed using ECL Western Blotting Substrate (Pierce). Proteins from cultures induced with 0, 0.02, and 0.2% L-arabinose (L-ara) are shown (lanes 1–3). The rHepP bands are indicated; the molecular weight standards are indicated in kDa by bars to the left of the blot. c rHepP exhibits heparinase activity. An agar plate containing 1 mg/mL porcine intestinal heparin in 1.5% agarose at pH 7 was spotted with 28 μg of rHepP in 20 μL of protein elution buffer. As a positive control (+ Ctl), 0.25 U of heparinase III from P. heparinum was spotted on the agar; the protein buffer was used as a negative control (− Ctl). After 1 h of incubation at 37 °C, 2% protamine sulfate solution was poured on the entire plate. Following incubation at room temperature for 2 h, the plate was examined for clear zones that indicate the presence of active heparinase

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