Construction and preservation of a stable and highly expressed recombinant Helicobacter pylori vacuolating cytotoxin A with apoptotic activity

Abstract

Background: H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer.

Results: A 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA^34-854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay.

Conclusions: A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

Keywords: Apoptosis; Expression; Helicobacter pylori; Preservation; Purification; VacA recombinant protein.

Fig. 1

Successful construction of the recombinant plasmid. A. The construction diagram of a vacA carrier: pET-41b-vacA^34–854. Gene name: vacA^34–854; Gene length: 2502 bp; Carrier name: pET-41b; Enzyme cutting sites: NdeI + XhoI; C terminal integrates the 8*His label. B. pET-41b-vacA^34–854 enzyme cutting identification. M: 1 Kb DNA ladder; Lane 1: recombinant plasmid pET-41b-vacA^34–854 cut by NdeI & XhoI endonucleases. C. Positive clone sequencing of pET41b-VacA^34–854 recombinant plasmid

Fig. 2

Expression and purification of the VacA recombinant protein in the pET-41b-vacA^34–854- E. coli BL21(DE3) system. A and B The expression of the VacA recombinant protein in E. coli BL21(DE3) cells as detected by SDS-PAGE and western blotting, respectively. NC: no induced cell lysis; Lane 1: 15 °C in the induction of cell lysis of 16 h; and Lane 2: 15 °C in the induction of 16 h of cell lysis supernatant; Lane 3: 15 °C in precipitation of cell lysis induced 16 h. C VacA recombinant protein in the inclusion bodies purified with Ni column (IDA) as detected by SDS-PAGE analysis. Lane 1: Loading; Lane 2: Flow through; Lane 3: Elution with 20 mM imidazole; Lane 4: Elution with 500 mM imidazole; Lane 5: Ni resin after elution. Lane M: Protein marker. D and E VacA recombinant protein in the supernatant purified with Ni column (NTA) as detected by SDS-PAGE and western blotting, respectively. The column equilibration buffer consists of 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, and wash buffer consists of 50 mM Tris-HCl, 1% Triton-X114, 8 M urea, 150 mM Nacl, PH 8.0). The target protein was eluted using a stepwise gradient of imidazole (i.e. 20, 50, 100 and 500 mM) and the results are shown as below: Lane 1: Pellet of cell lysate after centrifugation; Lane 2: Supernatant of cell lysate after centrifugation; Lane 3: Flow through; Lane 3–5: Elution with 20 mM imidazole; Lane 6–7: Elution with 50 mM imidazole; Lane 8–10: Elution with 100 mM imidazole; Lane 11–13: Elution with 500 mM imidazole; Lane 14: Ni resin after elution. Lane M: Protein marker. PC:positive control

Fig. 3

Renaturation of the target recombinant protein by various buffers as detected by SDS-PAGE (left) and western blotting (right). Lane M: Protein marker; Lane 1: Protein before refolding; Lane 2: buffer 1 (50 mM Tris,10% Gly, 150 mM NaCl, pH 8); Lane 3: buffer 2 (50 mM Tris,10% Gly, 150 mM NaCl, 0.1 mM DL-dithiothreitol (DTT), pH 8.0); Lane 4: buffer 3 (1× phosphate buffered saline (PBS), pH 7.4; Lane 5: buffer 4 (1× PBS, 10% GLy, 500 mM NaCl, pH 7.4); Lane 6: buffer 5 (1 × PBS,10% GLy, 500 mM NaCl, 0.1 mM DTT, pH 7.4); Lane 7: buffer 6 (20 mM Tris, 2 M urea, 400 mM Arg, 2.5 mM cysteamine, 0.25 mM cystamine, pH 8.5); Lane 8: buffer 7 (50 mM acetic acid, pH 2.9)

Fig. 4

Expression of VacA recombinant protein in the supernatant and pellet as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. A and B Expression of VacA recombinant protein in the supernatant as detected by SDS-PAGE (left) and western blotting (right); C and D Expression of VacA recombinant protein in the pellet as detected by SDS-PAGE (left) and western blotting (right). BSA: bovine serum albumin(2.00 μg); R: VacA^34–854 (2.00 μg); Both M1 and M2 are protein markers

Fig. 5

Morphological changes of GES-1 cells as observed by inverted microscopy after incubation with a high final concentration (65 μg/mL) of VacA recombinant protein for 24 h. Cellular morphological changes were visualized before incubation (0 h) and 6, 12 and 24 h after incubation by invert microscopy (Magnification × 100). After 24 h stimulation of VacA recombinant protein (as indicated by the arrow), significant cell morphological changes evident of apoptosis including early nuclear immobilization and clustering in GES-1 cells were observed. A large number of nuclei were retracted and chromatin in the nucleus converged to the edge

Fig. 6

Morphological changes of GES-1 cells as demonstrated by the observed by transmission electron microscopy after incubation at a high final concentration (65 μg/mL) of VacA recombinant protein for 24 h. A The blank control (Magnification × 5000). B The buffer control (50 mM acetic acid, pH 2.9, Magnification × 5000). C VacA recombinant protein (at a final concentration of 65 μg/mL); C1: The cell volume is reduced and the surface microvilli disappears (Magnification × 5000); C2: Nucleus shrinks and agglutinates to the periphery (Magnification × 10,000); and C3 & C4: Cytoplasmic disintegration and vacuoles (Magnification × 10,000)

Fig. 7

Detection of cells apoptosis induced by VacA recombinant protein using TUNEL assay. VacA recombinant protein induced GES-1 cell apoptosis. GES-1 cells treated with VacA recombinant protein at a high concentration (at a final 65 μg/mL) for 24 h. A Representative images of the TUNEL assay (Magnification × 400). Nuclei were counterstained with DAPI (blue) and TUNEL-positive cells (red) indicate apoptosis. B Quantification of results. Results are presented as the mean ± standard deviation, ***: P < 0.001 vs. the buffer control group