Heterologous expression, purification, and immunological reactivity of a recombinant HSP60 from Paracoccidioides brasiliensis

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.

FIG. 1.

SDS-PAGE and immunoblot analysis of the recombinant P. brasiliensis HSP60. E. coli XL1 Blue cells harboring the pGEX-4T-3 plasmid were grown at 30°C to an A₆₀₀ of 0.6 and harvested before (lanes 1) and after (lanes 2) a 2-h incubation with 0.1 mM IPTG. The cells were concentrated by centrifugation and lysed by extensive sonication. After centrifugation, the supernatant was absorbed to a glutathione-Sepharose affinity column in the presence of thrombin for 16 h. The eluate was analyzed (lanes 2). (A) SDS-PAGE analysis. Lane 1, 25 μg of total protein; lane 2, 6 μg of purified recombinant HSP60. (B) Reaction to the monoclonal anti-HSP60 antibody. Lane 1, 25 μg of total protein; lane 2, 500 ng of purified HSP60. The arrows indicate the HSP60 recombinant protein.

FIG. 2.

Reactivities of individual serum samples from P. brasiliensis-infected patients to the recombinant HSP60 as determined by Western blotting. Recombinant purified HSP60 (2.5 μg) was fractionated on an SDS-15% polyacrylamide gel and transferred to a nitrocellulose membrane. (A and B) Reactivity to serum samples (at a 1:500 dilution) from P. brasiliensis-infected patients with chronic and acute disease, respectively. (C) Reactivity of human control sera. Alkaline phosphatase-conjugated anti-human IgG antibody was used at a dilution of 1:1,000. The reactions were developed with BCIP-NBT. Lanes 1, reaction of the purified P. brasiliensis HSP60 to the monoclonal antibody. Lanes 2 to 12, reaction to individual sera at a 1:500 dilution. The numbers on the left are molecular masses in kilodaltons. The arrows indicate the relative gel migration of the purified recombinant HSP60.

FIG. 3.

Reactivities of sera from individuals with different diseases to the recombinant P. brasiliensis HSP60. Purified recombinant protein HSP60 (2.5 μg) was fractionated by one-dimensional gel electrophoresis (SDS-15% PAGE) and transferred to nitrocellulose membranes. Reactivities of serum samples from individuals with histoplasmosis (lanes 2 to 4), aspergillosis (lanes 5 to 7), cryptococcosis (lanes 8 to 10), sporotrichosis (lanes 11 to 13), and tuberculosis (lanes 14 to 16) are shown. Lane 1, reaction to the monoclonal anti-HSP60 antibody. The arrow indicates the relative migration of the purified recombinant HSP60.