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GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
Published on: October 30, 2013
Heterologous expression, purification, and immunological reactivity of a recombinant HSP60 from Paracoccidioides
Daniela A Cunha1, Roseli M Zancopé-Oliveira, M Sueli
1Laboratório de Biologia Molecular, ICB, Universidade Federal de Goiás, 74001-970 Goiânia, Goiás, Brazil.
Abstract:
The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients.
