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Cellular immune responses to recombinant heat shock protein 70 from Histoplasma capsulatum.

ABSTRACT: Heat shock protein (hsp) 70 from several microbes is antigenic in mammals. In this study we sequenced and expressed the gene encoding this protein from Histoplasma capsulatum to study its immunological activity. The deduced amino acid sequence of the gene demonstrated 71 and 76% identity to hsp7O from humans and Saccharomyces cerevisiae, respectively. A cDNA was synthesized by reverse transcription-PCR and was expressed in Escherichia coli. Recombinant protein reacted with a mouse monoclonal antibody raised against human hsp7O. Splenocytes from C57BL/6 mice immunized with recombinant hsp7O emulsified in adjuvant, but not yeast cells, reacted in vitro to the antigen. Recombinant hsp7O elicited a cutaneous delayed-type hypersensitivity response in mice immunized with protein or with viable yeast cells. Mice were injected with recombinant hsp7O and challenged intranasally with a sublethal inoculum of yeast cells. Vaccination did not confer protection in this model. Thus, recombinant hsp7O can induce a cell-mediated immune response but does not induce a protective response.

SUBMITTER: Allendoerfer R

PROVIDER: S-EPMC174346 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

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Cellular immune responses to recombinant heat shock protein 70 from Histoplasma capsulatum.

Allendoerfer R R Maresca B B Deepe G S GS

Infection and immunity 19961001 10

Heat shock protein (hsp) 70 from several microbes is antigenic in mammals. In this study we sequenced and expressed the gene encoding this protein from Histoplasma capsulatum to study its immunological activity. The deduced amino acid sequence of the gene demonstrated 71 and 76% identity to hsp7O from humans and Saccharomyces cerevisiae, respectively. A cDNA was synthesized by reverse transcription-PCR and was expressed in Escherichia coli. Recombinant protein reacted with a mouse monoclonal ant ...[more]

PMID: 8926078

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Project description:Recombinant Escherichia coli cultures are used to manufacture numerous therapeutic proteins and industrial enzymes, where many of these processes use elevated temperatures to induce recombinant protein production. The heat-shock response in wild-type E. coli has been well studied. In this study, the transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a dual heat-shock recombinant protein induction were examined. Most classical heat-shock protein genes were identified as regulated in both conditions. The major transcriptome differences between the recombinant and reported wild-type cultures were heavily populated by hypothetical and putative genes, which indicates recombinant cultures utilize many unique genes to respond to a heat-shock. Comparison of the dual stressed culture data with literature recombinant protein induced culture data revealed numerous differences. The dual stressed response encompassed three major response patterns: induced-like, in-between, and greater than either individual stress response. Also, there were no genes that only responded to the dual stress. The most interesting difference between the dual stressed and induced cultures was the amino acid-tRNA gene levels. The amino acid-tRNA genes were elevated for the dual cultures compared to the induced cultures. Since tRNAs facilitate protein synthesis via translation, this observed increase in amino acid-tRNA transcriptome levels, in concert with elevated heat-shock chaperones, might account for improved productivities often observed for thermo-inducible systems. Most importantly, the response of the recombinant cultures to a heat-shock was more profound than wild-type cultures, and further, the response to recombinant protein induction was not a simple additive response of the individual stresses. The objective of the present work is to gain a better understanding of the heat-shock response in recombinant cultures and how this response might impact recombinant protein production. To accomplish this objective, the transcriptome response of recombinant cultures subjected to a heat-shock and a dual heat-shock recombinant protein induction were analyzed. The transcriptome levels were determined using Affymetrix E. coli Antisense DNA microarrays, such that the entire genome was evaluated. These two transcriptome responses were also compared to recombinant cultures at normal growth temperature that were not over-expressing the recombinant protein and a set of literature recombinant culture data that were chemically induced to over-express the recombinant protein. Additionally, the heat-shock response of the recombinant cultures was compared to the literature report of the heat-shock response in wild-type cultures. The results of the global transcriptome analysis demonstrated that recombinant cultures respond differently to a heat-shock stress than wild-type cultures, where the transcriptome response of the recombinant cultures is further modified by production of a recombinant protein.

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Cellular immune responses to recombinant heat shock protein 70 from Histoplasma capsulatum.

Publications

Cellular immune responses to recombinant heat shock protein 70 from Histoplasma capsulatum.

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