Identification and cloning of an aspartyl proteinase from Coccidioides immitis.
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.
Johnson
S M
SM
Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis 95616, USA. smjohnson@ucdavis.edu
Kerekes
K M
KM
Zimmermann
C R
CR
Williams
R H
RH
Pappagianis
D
D
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003045:Coccidioides / Q000201:enzymology* / Q000235:genetics; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005657:Fungal Vaccines / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D005657:Fungal Vaccines; D011993:Recombinant Fusion Proteins; D016282:Aspartic Acid Endopeptidases