Expression and Single-step Purification of GRA8 Antigen of Toxoplasma gondii in Escherichia coli

Abstract

Diagnosis of Toxoplasma gondii (T.gondii) infection is of great medical importance especially for pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several toxoplasma antigens, including dense granule antigens (GRAs) has a great potential as diagnostic reagents. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags. In the present study, we produced truncated GRA8 (GRA8), excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coli (E.coli). GRA8 was purified using an optimized single-step Immobilized Metal ion Affinity Chromatography (IMAC). The purity and yield of GRA8 was highest at pH = 9.25. At this pH, 13.6 mg of GRA8 was obtained with the purity of 97.97%. Immunogenicity of the protein was evaluated in Western blot analysis showing the serum sample from a rabbit immunized with GRA8 recognized a single antigen of T.gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute toxoplasma infection in pregnant women, an indirect immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed using GRA8 resulting in a test specificity and sensitivity of 97.1% and 60.6%, respectively. These results demonstrated that immunogenic GRA8 can be produced in fusion with a short tag and purified near to homogeneity using an optimized IMAC. GRA8-IgM-ELISA was useful for detection of acute toxoplasma infection.

Keywords: Enzyme-linked immunosorbent assay; GRA8 protein; Gene expression; Immunoglobulin M; Toxoplasma gondii.

Figure 1

SDS-PAGE analysis of expression of TGRA8 in E.coli. A) Expression level of TGRA8 in three different E.coli strains. pET-28b(+)-TGRA8 construct was transformed into the E.coli BL21(DE3), BL21(DE3) plysS and Rosetta(DE3) cells and expression of TGRA8 was induced by addition of 0.1 mM IPTG. SDS-PAGE analysis showed the expression of an approximately 27 kDa protein in induced bacteria (AI) which was absent in uninduced bacteria (BI). The expression level of the recombinant protein seemed to be equal in the three bacterial strains. B) Expression level and solubility of TGRA8 in three different culture media. Recombinant Rosetta(DE3) bacteria were cultured in LB, TB and 2XTY culture media and expression of TGRA8 was induced. SDS-PAGE analysis showed that the highest expression level was achieved in cells cultured in LB medium. The soluble and insoluble fractions of induced bacteria were analyzed on SDS-PAGE. Most of the recombinant protein was in insoluble form. However, considerable amount of the protein cultured in LB was soluble. BI) Total proteins of E.coli cells harbouring pET-28b(+)-TGRA8 before induction. AI) Total proteins after induction with 0.1 mM IPTG.

Figure 2

Purification of TGRA8. A) Chromatograms of the immobilized metal affinity chromatography (IMAC) purification of TGRA8. The purification was performed by using the HisTrap FF column (5 ml) and buffers made at four pH values of 7.5, 8.1, 8.75 and 9.25. Initially, the column was equilibrated with the binding buffer (5 mM imidazole, 50 mM Tris–HCl, 500 mM NaCl). The soluble E.coli lysate was passed though the column and the unbound proteins were collected (Flow Through). The column was then washed step-wised with the binding buffer containing 35 (Wash 1), 60 (Wash 2) and 90 (Wash 3) mM imidazole. Absorbed material was eluted with the binding buffer containing 500 mM imidazole (Elution). The purity and yield of TGRA8 was improved when pH was increased from 7.5 to 9.25. The star mark possibly represents shows the peak of impurities. FT) flow through, W1: wash 1, W2: wash 2, W3: wash 3 and E: Elution. B) SDS-PAGE analysis of TGRA8 purified by IMAC at four different pH. Protein samples of unbound proteins (FT), wash 1 (W) and elution (E) fractions were analyzed. The purity of TGRA8 was considerably improved with increasing the pH from 7.5 to 9.25. The arrows represent bacterial proteins co-purified with TGRA8. The 54 kDa protein band, marked with a star sign, might be the dimmer of TGRA8 as it reacts with Toxoplasma-specific antibody on 12 % SDS-PAGE stained with Coomassie Blue. C) Silver nitrate staining of the purified TGRA8. At pH 9.25 TGRA8 was purified near to homogeneity. On silver stained gel only 2 specific bands of 27 and 54 kDa could be observed. M: Protein molecular weight marker

Figure 3

Western blotting analyses of purified TGRA8. A) Reactivity of purified TGRA8 with human sera. Purified TGRA8 was analyzed on the SDS-PAGE, transferred onto the nitrocellulose membrane and probed with the acute and chronic pooled sera from pregnant women. The purified antigen strongly reacted with IgG and IgM antibodies in acute sera, while no specific band can be observed on the blot probed with chronic sera,. The protein band of about 54 kDa was probably attributed to the dimmer of TGRA8. B) Evaluation of immunogenicity of TGRA8. One Rabbit was injected intramuscularly with 100 μg of TGRA8 three times with two weeks intervals. Two weeks after the last injection rabbit serum sample was obtained. Tachyzoites of T.gondii RH strain (5×10⁶ tachyzoites) were analyzed by SDS-PAGE, transferred onto nitrocellulose membrane and probed with the serum samples from the TGRA8-immunized and adjuvant-injected rabbits. The serum from the TGRA8-immunized rabbit recognized a single protein band of 35 kDa, the expected size of the native GRA8 antigen, while the serum from the adjuvant-injected rabbit failed to react with any specific protein of RH tachyzoites