Purification of the Recombinant Protein TmpA from Treponema pallidum Using Immobilized Metal Ion Affinity Chromatography, and Its Use in the Serodiagnosis of Syphilis

• Published in: Biotecnología aplicada Vol. 17; no. 2; pp. 89 - 93
• Main Authors: Izquierdo, Maricela, Silva, Eladio, Díaz, Héctor, Lubián, Ana L, Nibot, Carmen, Tabares, Dolores
• Format: Journal Article
• Language: English
• Published: Cuba Elfos Scientiae 31.12.2000
• Subjects: affinity chromatography; cromatografía de afinidad; diagnosis; diagnóstico; IMAC; protein purification; syphilis; sífilis; TmpA; TmpA protein; Treponema pallidum
• ISSN: 0864-4551

Two variants of the Treponema pallidum membrane protein A (TmpA-His, and TmpA) were expressed in Escherichia coli W3110 strain. TmpA-His was cloned with a six histidine tag at the C-terminus and was purified by immobilized metal ion affinity chromatography (IMAC). For this purpose, Sepharose 4B-IDA was used as matrix, and Ni 2+ and Cu2+ as chelating metal ions. Using this rapid and single-step chromatography the initial 20% purity of TmpA-His reached 90%. The overall yield was 70% and the binding capacity of the matrix for TmpA-His was approximately 1 mg/mL. TmpA was purified by electroelution and the yield was estimated to be 1 mg per each electroelution. The use of both proteins in the diagnosis of syphilis was evaluated by ELISA using panels of positive and negative sera. With respect to the serologic performance no differences between both proteins were found, which suggests the potential use of TmpA-His in the development of diagnostic systems for syphilis serodiagnosis.