title: 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.
authors: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
authors: Jalal
authors: J
authors: Babaie
authors: Mandana
authors: M
authors: Miri
authors: Ghazaleh
authors: G
authors: Sadeghiani
authors: Mehrak
authors: M
authors: Zare
authors: Ghader
authors: G
authors: Khalili
authors: Majid
authors: M
authors: Golkar
keywords: Enzyme-linked immunosorbent assay; GRA8 protein; Gene expression; Immunoglobulin M; Toxoplasma gondii