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Identification and cloning of an aspartyl proteinase from Coccidioides immitis
S M Johnson1, K M Kerekes, C R Zimmermann
1Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis 95616, USA. smjohnson@ucdavis.edu
Abstract:
A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.
Insights
Researchers isolated a 45 kDa protein from Coccidioides immitis, identified it as a fungal aspartyl proteinase, and cloned its gene. This recombinant protein will be tested for its potential as a vaccine candidate.
Area of Science:
- Mycology
- Molecular Biology
- Vaccinology
Background:
- Coccidioides immitis is a fungus causing coccidioidomycosis.
- Aspartyl proteinases are crucial enzymes in fungal pathogenesis.
- Identifying novel antigens is key for developing effective vaccines.
Purpose of the Study:
- To isolate and characterize a 45 kDa protein from Coccidioides immitis.
- To clone and express the gene encoding this protein.
- To prepare a recombinant protein for evaluating its immunogenicity.
Main Methods:
- Protein isolation from formaldehyde-killed spherules.
- N-terminal amino acid sequencing and homology analysis.
- Polymerase chain reaction (PCR) amplification and cDNA sequencing.
- Cloning, expression in E. coli, and purification of recombinant protein.
Main Results:
- A 45 kDa protein was isolated and identified as a fungal aspartyl proteinase.
- The cDNA encoding the proteinase was successfully amplified, cloned, and sequenced.
- A recombinant aspartyl proteinase with a C-terminal His-tag was expressed and purified.
Conclusions:
- A recombinant Coccidioides immitis aspartyl proteinase has been produced.
- This recombinant protein is a candidate for further studies on antigenicity and protective immunogenicity.
- The findings pave the way for potential vaccine development against coccidioidomycosis.