title: Expression, purification and improved antigenicity of the Mycobacterium tuberculosis PstS1 antigen for serodiagnosis.
abstract: The phosphate-specific transport substrate binding protein-1 (PstS1) is a potential antigen used for the serological diagnosis of tuberculosis. For a highly specific diagnostic result, it is important that the recombinant PstS1 be highly pure and correctly folded. In this study, the PstS1 was expressed as fusion protein with glutathione-S-transferase (PstS1-GST) and Escherichia coli trigger factor (PstS1-TF) and their immunodiagnostic potentials were evaluated. The insoluble PstS1-GST was denatured and refolded to the native conformation by a step-gradient dilution, followed by purification with affinity chromatography on immobilized glutathione whereas the soluble PstS1-TF was directly purified by Ni-NTA affinity and size-exclusion chromatographies. The levels of antibody responses to PstS1-TF and PstS1-GST were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of 22 tuberculosis patients with smear-positive and culture-positive tuberculosis as well as 20 healthy individuals; the antigenicities of the samples were evaluated in terms of sensitivity and specificity. To determine the diagnostic accuracy, receiver operation characteristic (ROC) curves were constructed and then the areas under the ROC curves (AUC) were calculated; the AUC values for PstS1-TF and PstS1-GST were 0.971 and 0.877 with 95% confidence intervals (CI) of 0.927-1.000 and 0.768-0.986, respectively. The specificity of PstS1-TF was reduced from 89.5% to 84.2%, but in case of PstS1-GST it dropped drastically from 78.9% to 26.3% when the sensitivity was raised from 86.4% up to 95.5%. These results indicate that PstS1-TF is capable of producing more accurate and consistent serodiagnostic results than PstS1-GST, possibly due to its conformation being closer to the native state.
authors: Department of Biological Sciences, Dankook University, Dandae-ro 119, Dongnam-gu, Cheonan-si, Chungnam 330-714, Republic of Korea.
authors: Won-Hyun
authors: WH
authors: Hwang
authors: Institute of Molecular Recognition and Nano Technology, Kookmin University, 861-2, Jeongneung-dong, Songbuk-gu, Seoul 136-702, Republic of Korea.
authors: Won-Kyu
authors: WK
authors: Lee
authors: Department of Research and Development, Korea Institute of Tuberculosis, 482, Mansuri, Osong, Cheongwon-gun, Chungbuk 363-954, Republic of Korea.
authors: Sung Weon
authors: SW
authors: Ryoo
authors: Department of Biological Sciences, Dankook University, Dandae-ro 119, Dongnam-gu, Cheonan-si, Chungnam 330-714, Republic of Korea.
authors: Ki-Yeol
authors: KY
authors: Yoo
authors: Department of Biological Sciences, Dankook University, Dandae-ro 119, Dongnam-gu, Cheonan-si, Chungnam 330-714, Republic of Korea. Electronic address: gtae@dankook.ac.kr.
authors: Gun-Sik
authors: GS
authors: Tae
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001431:Bacteriological Techniques / Q000379:methods*; D016022:Case-Control Studies; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D012372:ROC Curve; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D012698:Serologic Tests / Q000379:methods*; D012995:Solubility; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology
chemical_list: D018528:ATP-Binding Cassette Transporters; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C581205:PstS-1 protein, Mycobacterium tuberculosis; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase
keywords: Antigenicity; Mycobacterium tuberculosis; PstS1; ROC curve; Solubility