Identification and cloning of an aspartyl proteinase from Coccidioides immitis

A 45 kDa protein was isolated from a soluble vaccine prepared from formalde hyde-killed spherules of Coccidioides immitis. From the N-terminal amino ac id sequence, the protein yielded a 17-amino-acid peptide that was homologou s to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designe d from the 45 kDa N-terminal amino acid sequence and a Fungal aspartyl prot einase consensus amino acid sequence. The PCR product was cloned and sequen ced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cl oned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-termin al His-tag expressed in E. coli. The recombinant aspartyl proteinase was pu rified by immobilized metal affinity chromatography. This recombinant prote in will be used for further studies to evaluate its antigenicity, including protective immunogenicity. (C) 2000 Elsevier Science B.V. All rights reser ved.