One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Human blood was collected from healthy volunteers with prior written informed consent and approval from the Institutional Review Board of the National Tsing Hua University, Hsinchu, Taiwan.

1. Expression of Recombinant HP-NAP in E. coli

1. Prepare the plasmid pET42a-NAP containing the DNA sequence of HP-NAP from H. pylori 26695 strain as previously described¹⁶. Prepare the plasmids containing the DNA sequence of HP-NAP with the desired point mutations as described (see Protocol, step 8).
2. Transform 10 ng of the above DNA plasmids into E. coli BL21 (DE3) competent cells by heat shock for 45 seconds at 42 °C, streak the cells on lysogeny broth (LB) agar plates containing 50 µg/ml kanamycin, and incubate the plates at 37 °C for 16 hr.
3. Inoculate single colonies in individual tubes containing 5 ml of LB broth with 50 µg/ml kanamycin and grow them as precultures at 37 °C for 16 hr with shaking at 170 rpm.
4. Inoculate 2 ml of the above pre-culture cells into 200 ml of LB broth containing 50 µg/ml kanamycin in a 1 L flask. Incubate the inoculated culture flask at 37 °C for 2 hr with shaking at 170 rpm until the absorbance at 600 nm reaches approximately 0.4-0.5 detected by an UV/VIS spectrophotometer.
5. Add 80 µl of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) into the above culture to a final concentration of 0.4 mM to induce the expression of HP-NAP. Incubate the culture for 3 hr until the absorbance at 600 nm reaches approximately 1.6-1.7.
6. Centrifuge the cells at 6,000 x g at 4 °C for 15 min to remove the supernatant. Store the cell pellet at −70 °C until purification.

2. Preparation of the Soluble Protein Fraction Containing HP-NAP

Note: All of the following steps are carried out at 4 °C.

1. Resuspend 50 ml of the cell pellet prepared in Protocol step 1.6 in 20 ml of 20 mM Tris-HCl, pH 9.0, 50 mM NaCl containing 0.13 mM phenylmethylsulfonyl fluoride (PMSF), 0.03 mM N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK), and 0.03 mM N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) at 4 °C as previously described¹⁶.
2. Disrupt the bacteria cells by passing the bacterial suspension through a high pressure homogenizer operated at a range of 15,000 to 20,000 psi for 7 times at 4 °C.
3. Centrifuge the cell lysate at 30,000 × g at 4 °C for 1 hr to separate the soluble and insoluble protein fractions by using an ultracentrifuge. Transfer the supernatant as the soluble protein fraction to a beaker.
4. Measure the protein concentration of the soluble protein fraction by the Bradford method using a commercial kit with bovine serum albumin (BSA) as the standard according to the manufacturer's instructions.
5. Add 177.6 µl of 1 N HCl into 20 ml of the soluble protein fraction obtained from Protocol step 2.3 to adjust its pH value from pH 9.0 to pH 8.0.
6. Add 20 mM Tris-HCl, pH 8.0, 50 mM NaCl to the above protein solution to make the final protein concentration to be 0.5 mg/ml.

3. Purification of Recombinant HP-NAP From E. coli by Negative Mode Batch Chromatography with DEAE Ion-exchange Resins

1. Prepare 15 ml of DEAE resins.
   1. Weigh out 0.6 g dry powder of DEAE resins and suspend it in 30 ml of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl at room temperature for at least 1 day.
   2. Centrifuge the resin at 10,000 x g at 4 °C for 1 min.
   3. Remove and discard the supernatant.
   4. Add 15 ml of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl.
   5. Repeat Protocol steps 3.1.2 to 3.1.4 four more times.
   6. Store the 15 ml settled resin (50% slurry in 30 ml Tris buffer) at 4 °C for subsequent use.
      Note: All of the following steps are carried out at 4 °C.
2. Add 45 ml of the soluble proteins prepared in Protocol step 2.6 to 15 ml of the resin and stir the protein/resin slurry with a magnetic stirrer at 4 °C for 1 hr.
3. Pour the protein/resin slurry into a plastic or glass column fitted with a stopcock. Allow the resin to settle under gravity.
4. Open the stopcock to allow the protein solution to run through the column by gravity flow until the liquid level in the column is just above the resin. Collect the flow-through as the unbound fraction, which contains the purified HP-NAP.
5. Add 15 ml of ice-cold 20 mM Tris-HCl, pH 8.0, 50 mM NaCl into the column.
6. Open the stopcock to allow the wash buffer to run through the column by gravity flow until the liquid level in the column is just above the resin. Collect the flow-through as the wash fraction.
7. Repeat Protocol steps 3.5 to 3.6 four more times to collect the additional wash fractions.
8. Add 15 ml of ice-cold 20 mM Tris-HCl, pH 8.0, 1 M NaCl into a column.
9. Open the stopcock to allow the elution buffer to run through the column by gravity flow until the liquid level in the column is just above the resin. Collect the flow-through as the elution fraction.
10. Repeat Protocol steps 3.8 to 3.9 four more times to collect the additional elution fractions.

4. Buffer Exchange and Endotoxin Removal of HP-NAP Purified by Negative Mode Batch Chromatography with DEAE Resins

Note: The purified HP-NAP expressed in E. coli needs to be subjected to buffer exchange and endotoxin removal prior to stimulate neutrophils.

1. Perform dialysis to change the buffer of the purified HP-NAP to Dulbecco's phosphate-buffered saline (D-PBS), pH 7.2, at 4 °C by using dialysis tubing with molecular weight cutoff of 14 kDa as previously described²⁴.
2. Filter the dialyzed HP-NAP through a syringe filter with a positively-charged, hydrophilic membrane attached to a 20 ml disposable syringe at flow rates ranging from 2.5 to 4 ml/minute at 4 °C to remove endotoxin.

5. Characterization of the Molecular Properties of Purified Recombinant HP-NAP by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western Blotting, Native-PAGE, Gel Filtration Chromatography, and Circular Dichroism Spectroscopy

1. Analyze the purified recombinant HP-NAP by SDS-PAGE and western blotting with hybridoma culture supernatants containing mouse monoclonal antibody MAb 16F4²⁵ as previously described²⁴.
2. Analyze the purified HP-NAP by native-PAGE and gel filtration chromatography to examine its oligomeric status as previously described²⁶.
3. Analyze the purified recombinant HP-NAP by circular dichroism spectroscopy to examine its secondary structure as previously described²⁶.

6. Evaluation of the Purity of HP-NAP by Silver Staining of a SDS-PAGE Gel

1. Prepare the fixing solution, sensitizing solution, silver solution, developing solution, stop solution, and washing solution according to the manufacturer's instructions of a silver staining kit.
2. Perform silver staining of a SDS-PAGE gel according to the manufacturer's instructions of a silver staining kit.
3. Acquire the image of the gel with an imaging system.

7. Measurement of ROS Production From Neutrophils Induced by HP-NAP

1. Isolate human neutrophils from human heparinized blood by dextran sedimentation followed by density gradient centrifugation as previously described²⁴.
2. Measurement of ROS production from neutrophils stimulated with HP-NAP by using a luminol-dependent chemiluminescence assay.
   1. Turn on a plate reader and set the temperature at 37 °C. Place an empty flat bottom 96-well white plate inside the plate reader chamber, allowing it to warm to 37 °C.
   2. Prepare the stimulus mixtures in the D-PBS, pH 7.2, containing 0.9 mM CaCl₂, 0.5 mM MgCl₂, and 13.3 µM 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) with the presence and absence of 0.67 µM endotoxin-removed HP-NAP prepared from Protocol step 4.2.
   3. Adjust the concentration of human neutrophils prepared from Protocol step 7.1 to 2 x 10⁶ cells/ml in D-PBS, pH 7.2, containing 5 mM glucose.
   4. Add 50 µl of neutrophil suspension into each well of the 96-well while plate.
   5. Add 150 µl of the stimulus mixtures prepared from Protocol step 7.2.2 into the well containing neutrophils at a time interval of 5 seconds per well.
   6. Measure the emission of chemiluminescence for 5 sec per well over a period of three hours by using a plate reader.

8. Construction of DNA Plasmids Harboring HP-NAP Mutants by Polymerase Chain Reaction (PCR)-based Site-direct Mutagenesis

Note: PCR-based site-direct mutagenesis was generated basically as described previously²⁷ except that the "silent" restriction sites were introduced to the mutagenesis primers by site-directed mutagenesis (SDM)-assist software²⁸.

1. Perform PCR Reaction.
   1. Add 10 ng of plasmid pET42a-NAP, 1 µM of mutagenesis primer pairs listed in Table 1, 200 µM of deoxynucleoside triphosphates (dNTPs), and 3 units of high-fidelity PCR enzyme mix into a PCR tube containing deionized water, giving a final volume of 25 µl.
   2. Initiate the PCR cycles at 95 °C for 10 min to denature the template DNA, and follow with 12 amplification cycles at 95 °C for 1 minute, T_{m no}-5 °C for 1 minute, and 72 ^oC for 6 min.
   3. Finish the PCR cycles with an annealing step at T_{m pp}-5 ^oC for 1 minute and an extension step at 72 °C for 30 min.
2. Treat 15 µl of the PCR product with 0.4 µl of Dpn I restriction enzyme at 37 °C for 2 hr and then analyze 2 µl of the Dpn I-treated PCR product by agarose gel electrophoresis.
3. Screen mutants.
   1. Transform 2 µl of the PCR product into E. coli DH5α competent cells by heat shock for 45 sec at 42 °C.
   2. Spread the transformed cells on a LB plate containing 50 µg/ml kanamycin and incubate the plate at 37 °C for 16 hr.
   3. Isolate the plasmid DNA from the bacterial colonies using a commercial alkaline lysis kit according to the manufacturer's protocol.
   4. Treat the plasmid DNA isolated in Protocol step 8.3.3 with XhoI restriction enzyme to verify the presence of the desired silent mutations according to manufacturer's protocol.
   5. Sequence the plasmid DNA verified by Protocol step 8.3.4 with T7 promoter primer to confirm the correction of the coding sequences of HP-NAP mutants according to manufacturer's protocol.