Identification and cloning of an aspartyl proteinase from Coccidioides immitis

Suzanne M. Johnson, Kaye M. Kerekes, C. Roger Zimmermann, Robert H. Williams, Demosthenes Pappagianis

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)213-222
Number of pages10
JournalGene
Volume241
Issue number2
DOIs
StatePublished - Jan 11 2000

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Keywords

  • Coccidioidomycosis
  • Enzyme
  • Fungus
  • Pathogen
  • Vaccine

ASJC Scopus subject areas

  • Genetics

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