Selective Purification of a Bacterial Protein by Negative Ion-Exchange Chromatography

Abstract

Source: Kuo, T., et al. One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode. J. Vis. Exp. (2016)

The video demonstrates a negative chromatography technique to purify a virulence-associated protein from a bacterial lysate. By using a positively charged resin in a buffered solution, host-cell proteins are selectively retained, while the near-neutral target protein remains unbound and is collected in the flow-through. This method allows efficient separation and recovery of the target protein for downstream applications.

Protocol

1. Expression of Recombinant HP-NAP in Escherichia coli (E. coli)

1. Prepare the plasmid pET42a-NAP containing the DNA sequence of Helicobacter pylori neutrophil-activating protein (HP-NAP) from H. pylori 26695 strain as previously described. Prepare the plasmids containing the DNA sequence of HP-NAP with the desired point mutations.
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 ultraviolet-visible (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 described.
2. Disrupt the bacterial 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 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 diethylaminoethyl (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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
pET42a-NAP	N/A	N/A	prepared as described in Supplementary data of Refernce 15 http://www.sciencedirect.com/science/article/pii/S0006291X08018317
E. coli BL21 (DE3)	Thermo Fisher Scientific Inc	C6000-03	https://www.thermofisher.com/order/catalog/product/C600003
Kanamycin	Amresco	25389-94-0	http://www.amresco-inc.com/KANAMYCIN-SULFATE-0408.cmsx
Isopropyl β-D-1-thiogalactopyranoside (IPTG)	MD Biomedical Inc	101-367-93-1	http://www.antibody-antibodies.com/product_det.php?id=238064&supplier=search&name=IPTG%20
Phenylmethylsulfonyl fluoride (PMSF)	Sigma-Aldrich	10837091001	protease inhibitor
N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK)	Sigma-Aldrich	T7254	protease inhibitor http://www.sigmaaldrich.com/catalog/product/sigma/t7254?lang=en®ion=TW
N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK)	Sigma-Aldrich	T4376	protease inhibitor http://www.sigmaaldrich.com/catalog/product/sigma/t4376?lang=en®ion=TW
Protein standard (bovine serum albumin)	Sigma-Aldrich	P5619	a standard protein for Bio-Rad Protein Assay http://www.sigmaaldrich.com/catalog/product/fluka/p5619?lang=en®ion=TW
DEAE–Sephadex A-25 chloride form	Sigma-Aldrich	A25120	http://www.sigmaaldrich.com/catalog/product/sigma/a25120?lang=en®ion=TW
Spectrum/Por dialysis tubing	Spectrum Laboratories	132720	with molecular weight cutoff of 14 kDa http://www.spectrumlabs.com/dialysis/RCtubing.html?Pn=132720;
E. coli DH5α	Thermo Fisher Scientific Inc	18265-017	https://www.thermofisher.com/order/catalog/product/18265017
U-2800 double beam UV/VIS spectrophotometer	Hitachi	N/A	out of market and upgraded to a new model http://hitachi-hta.com/products/life-sciences-chemical-analysis/uvvisible-spectrophotometers
EmulsiFlex-C3 high pressure homogenizer	Avestin Inc	C315320	http://www.avestin.com/English/c3page.html
Hitachi Koki himac CP80WX general ultracentrifuge	Hitachi Koki Co	90106401	for separation of the soluble and insoluble protein fractions from E. coli lysates http://centrifuges.hitachi-koki.com/products/ultra/cp_wx/cp_wx.html