Fig 1.
SDS-PAGE analysis of the purification of HP-NAP by DEAE negative mode chromatography at pH 7.0 to 9.0.
The soluble protein fraction of E. coli BL21(DE3) expressing HP-NAP was adjusted to pH ranging from 7.0 to 9.0 at 25°C and then diluted to a protein concentration of 0.3 mg/ml. The samples were individually loaded onto DEAE Sepharose and DEAE Sephadex resins to purify recombinant HP-NAP by a batch method as described in Materials and Methods. The pH-adjusted soluble protein fractions, indicated as load, and the unbound, wash and elution fractions were analyzed by SDS-PAGE. Molecular masses (M) in kDa are indicated on the left of the stained gels and the blots. The percent ratio of the amount of recombinant HP-NAP detected in each fraction to the amount of HP-NAP loaded on the resin at each pH was calculated from the intensity of HP-NAP band on SDS gels for each fraction divided by the sum of those for the unbound, wash and elution fractions. Similar results were obtained from at least two independent experiments.
Fig 2.
Immunoblot and native‐PAGE analysis of the purification of HP‐NAP by DEAE negative mode chromatography at pH 7.0 to 9.0.
The protein samples are prepared the same as those described in Fig 1. The pH-adjusted soluble protein fractions, indicated as load, and the unbound, wash and elution fractions collected from DEAE Sephadex and DEAE Sepharose resins were analyzed by immunoblotting (A) and native‐PAGE (B). Molecular masses (M) in kDa are indicated on the left of the stained gels. Similar results were obtained from at least two independent experiments.
Fig 3.
Schematic diagram of experimental approaches to investigate the effect of the surface charge of HP-NAP and impure proteins on the interaction of HP-NAP and DEAE resins.
Fig 4.
Binding ability of HP-NAP purified from DEAE negative mode chromatography to DEAE resins at pH 7.0 to 9.0.
The purified recombinant HP-NAP obtained from the unbound fraction of DEAE Sepharose and DEAE Sephadex resins from the batch chromatography at pH 7.0 to 9.0 at 25°C was re-subjected to the batch chromatography under the same conditions as described in Materials and Methods to examine the binding ability of pure HP-NAP to the two DEAE resins. The unbound (U), wash (W) and elution (E) fractions were analyzed by SDS-PAGE on 15% gels. Molecular masses (M) in kDa are indicated on the stained gels. The percent ratio of the amount of recombinant HP-NAP detected in each fraction to the amount of HP-NAP loaded on the resin at each pH was calculated from the intensity of HP-NAP band on SDS gels for each fraction divided by the sum of those for the unbound, wash and elution fractions. Similar results were obtained from at least two to three independent experiments. Note: The purified recombinant HP-NAP obtained from the unbound fraction from DEAE negative mode chromatography at 9.0, indicated as load (L), is shown.
Fig 5.
Capillary electropherograms of HP-NAP purified from DEAE negative mode chromatography at pH 7.0 to 9.0.
The recombinant HP-NAP obtained from the unbound fractions of DEAE Sepharose and DEAE Sephadex resins at pH 7.0 to 9.0 was subjected to CE analysis at 25°C as described in Materials and Methods. The UV absorbance was recorded at 220 nm. 4-Methoxybenzyl alcohol (4mBA) was used as the neutral electroosmotic flow (EOF) marker, which migrated as a peak at μe = 0 for all electropherograms. The peaks shown in the electropherograms are HP-NAP (━), 4mBA (—-), and HP-NAP together with 4mBA (⋯). Data were representative of two independent experiments.
Table 1.
The proposed surface charge and the calculated electrophoretic mobility of HP-NAP purified by DEAE negative mode chromatography and gel-filtration chromatography at pH 7.0 to 9.0 based on the CE data.
Fig 6.
Purification of recombinant HP-NAP expressed in E. coli by gel-filtration chromatography.
HP-NAP expressed in E. coli BL21(DE3) was purified by two consecutive gel filtration steps using an XK 16/100 column packed with Sephacryl S-300 high resolution resin (Sephacryl S-300 HR) and a HiLoad 16/60 Superdex 200 prep grade (Superdex 200 pg) gel filtration column at pH 9.0 (25°C) as described in Materials and Methods. The chromatogram (A) of HP-NAP (━) eluted from Superdex 200 pg was recorded at UV absorbance of 280 nm. The molecular mass of each protein marker (—-) was indicated at the top of each peak shown in the chromatogram. Insoluble pellet (I), whole cell lysate (W), soluble cytoplasmic fraction (S), and the eluates from the two columns were analyzed by SDS-PAGE (B) and native-PAGE (C). Molecular masses (M) in kDa are indicated on the stained gels. Data were representative of three independent experiments.
Fig 7.
Analysis of molecular and structural properties of HP-NAP purified from gel-filtration chromatography at pH 7.0 to 9.0.
HP-NAP purified by gel-filtration chromatography at pH 9.0 (25°C) was adjusted to pH 8.5, 8.0, 7.5, and 7.0 as described in Materials and Methods. (A) Native-PAGE analysis of HP-NAP at the indicated pH at 25°C. The molecular masses (M) in kDa are indicated on the stained gels. Data were representative of three independent experiments. (B) Sedimentation coefficient of HP-NAP at the indicated pH monitored by analytical ultracentrifugation (AUC) at 25°C. Sedimentation coefficient distribution c(s) was shown as a function of S. The c(s) distribution was analyzed by using the software program SEDFIT. (C) The far-UV circular dichroism (CD) spectra of HP-NAP at the indicated pH at 25°C. The spectra were recorded at the wavelength range of 195 to 260 nm at 25°C.
Fig 8.
Binding ability of recombinant HP-NAP purified from gel-filtration chromatography to DEAE resins at pH 7.0 to 9.0.
HP-NAP purified by gel-filtration chromatography at pH 9.0 (25°C) was kept at pH 9.0 or adjusted to pH 8.5, 8.0, 7.5, and 7.0 and then diluted to a protein concentration of 0.3 mg/ml. The samples were then subjected to batch chromatography by using DEAE Sepharose and DEAE Sephadex resins at their respective pH at 25°C. The unbound (U), wash (W) and elution (E) fractions were analyzed by SDS-PAGE on 15% gels. Molecular masses (M) in kDa are indicated on the stained gels. The percent ratio of the amount of recombinant HP-NAP detected in each fraction to the amount of HP-NAP loaded on the resin at each pH was calculated from the intensity of HP-NAP band on SDS gels for each fraction divided by the sum of those for the unbound, wash and elution fractions. Similar results were obtained from two independent experiments.
Fig 9.
Capillary electropherograms of HP-NAP purified by gel-filtration chromatography at pH 8.5 and 9.0.
HP-NAP purified by gel-filtration chromatography at pH 9.0 (25°C) was kept at pH 9.0 or adjusted to pH 8.5 and then diluted to a protein concentration of 0.1 mg/ml. The CE analysis was performed at 25°C with UV absorbance recorded at 220 nm as described in Materials and Methods. The peaks shown in the electropherograms are HP-NAP (━), 4mBA (—-), and HP-NAP together with 4mBA (⋯). 4mBA, as the neutral EOF marker, was shown as a peak at μe = 0 for all electropherograms. Data were representative of two independent experiments.