Figure 1.
Recombinant HP-NAP expressed in B. subtilis as a soluble, multimeric protein.
B. subtilis DB104-pRPA-NAP and B. subtilis DB104 were incubated at 37°C for 15 hr. The cells were harvested, and the whole cell lysates were prepared as described in Materials and Methods. The whole cell lysate (W) from B. subtilis DB104-pRPA-NAP was further centrifuged to separate the soluble fraction (S) and insoluble pellet (I). The samples were analyzed by SDS-PAGE (A), immunoblotting (B) and native-PAGE (C). Recombinant HP-NAP purified from E. coli BL21(DE3) harboring pET42a-NAP was used as a positive control (P). Molecular weights (M) in kDa are indicated on the left of the stained gels and the blot.
Figure 2.
Optimization of pH values and DEAE resins for purification of recombinant HP-NAP expressed in B. subtilis.
The soluble fraction from the whole cell lysate of B. subtilis DB104-pRPA-NAP was adjusted to the indicated pH values to purify recombinant HP-NAP using DEAE Sephadex and DEAE Sepharose resins by a batch method as described in Materials and Methods. The soluble fraction from the whole cell lysate of B. subtilis DB104-pRPA-NAP, indicated as load, and the unbound supernatant, wash fraction, and elution fraction collected using DEAE Sephadex (A) and DEAE Sepharose (B) resins were analyzed by SDS-PAGE. Molecular weights (M) in kDa are indicated on the left of the stained gels.
Figure 3.
Optimization of the ratio of the amount of proteins loaded onto DEAE Sephadex resins for purification of recombinant HP-NAP expressed in B. subtilis.
The soluble fraction from the whole cell lysate of B. subtilis DB104-pRPA-NAP was adjusted to pH 8.0. This sample, indicated as load (L), was then loaded onto DEAE Sephadex resins according to the indicated ratio of mg proteins per milliliter of resins to purify recombinant HP-NAP by a batch method as described in Materials and Methods. The soluble fraction from the whole cell lysate of B. subtilis DB104-pRPA-NAP, the unbound supernatant, wash fraction, and elution fraction were analyzed by SDS-PAGE. Molecular weights (M) in kDa are indicated on the stained gels.
Figure 4.
Purification of recombinant HP-NAP from B. subtilis by DEAE Sephadex anion-exchange chromatography.
A, The soluble fraction from the whole cell lysate of B. subtilis DB104-pRPA-NAP was applied to DEAE Sephadex anion-exchange column as described in Materials and Methods. The chromatogram was recorded by UV absorbance at 280 nm. The fractions of flow-through, wash, and elution were ranged from fractions 1 to 22, 23 to 42, and 43 to 63, respectively. The inset represents the enlarged chromatogram of fractions 1 to 37. B, The whole cell lysate (W), soluble fraction (S), and insoluble pellet (I) of B. subtilis DB104-pRPA-NAP and the selected fractions corresponding to the fraction number of chromatogram shown in (A) were analyzed by SDS-PAGE. Molecular weights (M) in kDa are indicated on the left of the stained gels.
Figure 5.
PAGE and immunoblot analysis of the purification process of recombinant HP-NAP expressed in B. subtilis.
The whole cell lysate (W) and soluble fraction (S) of B. subtilis DB104-pRPA-NAP, HP-NAP-containing flow-through fractions (F) obtained by DEAE Sephadex chromatography, and the concentrated HP-NAP (C) from flow-through fractions were analyzed by SDS-PAGE (A), native-PAGE (B), and immunoblotting (C) with an anti-HP-NAP antibody (MAb 16F4). Molecular weights (M) in kDa are indicated on the left of stained gels and the blot.
Table 1.
Purification summary table of recombinant HP-NAP from B. subtilis.
Figure 6.
Gel-filtration chromatographic analysis of recombinant HP-NAP purified from B. subtilis.
Purified HP-NAP and protein molecular weight makers were subjected to gel-filtration chromatography as described in Materials and Methods. The chromatograms were recorded by UV absorbance at 280 nm. The molecular weight of each protein marker was indicated at the top of each peak shown in the chromatogram.
Figure 7.
Analyses of molecular and structural properties of recombinant HP-NAP purified from B. subtilis.
The molecular weight (A), sedimentation coefficient (B), and secondary structure (C) of HP-NAP were analyzed by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS), analytical ultracentrifugation, and circular dichroism spectroscopy, respectively, as described in Materials and Methods. A, The peak in mass spectrum is corresponding to the molecular weight of HP-NAP monomer. B, The sedimentation coefficient distribution c(S) is shown as a function of S. The c(S) distribution was analyzed using the software program SEDFIT. C, The far UV circular dichroism spectrum of HP-NAP was recorded at the wavelength range of 195 to 260 nm.
Table 2.
Comparison of the molecular properties of HP-NAP characterized from this and other studies.
Figure 8.
Production of reactive oxygen species in human neutophils induced by recombinant HP-NAP purified form B. subtilis.
A, Human neutrophils (1×105 cells) were treated with 1 µM HP-NAP, 0.08 µM phorbol 12-myristate 13-acetate (PMA) as a positive control, D-PBS and 0.05% DMSO in D-PBS as negative controls at 37°C for 4 hr. The contents of ROS generated in neutrophils were measured continuously by using a 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCF-DA)-dependent assay as described in Materials and Methods. The result was represented as the profile of one experiment in triplicate. B, The fluorescent intensities detected from human neutrophils treated with indicated stimuli for 1.5 hr as described in (A) are shown. Data were represented as the mean ± S.D. of six independent experiments in triplicate (*p<0.01).