Characterization of a cDNA encoding a subtilisin-like serine protease (NC-p65) of Neospora caninum

Kitland Louie, Patricia A Conrad

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The NC-p65 cDNA is the first protease sequence cloned and described for Neospora caninum. The full length cDNA was isolated by 5'- and 3'-rapid amplification of cDNA ends (RACE). NC-p65 was composed of 865 amino acids with a predicted signal sequence, a proposed pro-domain, and an internal region of conserved repeats. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) and had a predicted active site made up of the catalytic residues, Asp 253, His 309, and Ser 484. Antibodies to recombinant NC-p65 recognized multiple bands on Neospora lysate immunoblots, but most intensely stained a 65 kDa band. When N. caninum proteins were purified with affinity resins specific for NC-p65 and analyzed for enzyme activity, a single specific band of reaction was observed on gelatin- saturated zymograms.

Original languageEnglish (US)
Pages (from-to)211-223
Number of pages13
JournalMolecular and Biochemical Parasitology
Volume103
Issue number2
DOIs
StatePublished - Oct 15 1999

Fingerprint

Neospora
Subtilisin
Serine Proteases
Complementary DNA
Protein Sequence Analysis
Gelatin
Protein Sorting Signals
Catalytic Domain
Proteins
Peptide Hydrolases
Amino Acids
Antibodies
Enzymes

Keywords

  • NC-p65
  • Neospora caninum
  • Subtilase
  • Subtilisin-like serine protease

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Characterization of a cDNA encoding a subtilisin-like serine protease (NC-p65) of Neospora caninum. / Louie, Kitland; Conrad, Patricia A.

In: Molecular and Biochemical Parasitology, Vol. 103, No. 2, 15.10.1999, p. 211-223.

Research output: Contribution to journalArticle

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N2 - The NC-p65 cDNA is the first protease sequence cloned and described for Neospora caninum. The full length cDNA was isolated by 5'- and 3'-rapid amplification of cDNA ends (RACE). NC-p65 was composed of 865 amino acids with a predicted signal sequence, a proposed pro-domain, and an internal region of conserved repeats. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) and had a predicted active site made up of the catalytic residues, Asp 253, His 309, and Ser 484. Antibodies to recombinant NC-p65 recognized multiple bands on Neospora lysate immunoblots, but most intensely stained a 65 kDa band. When N. caninum proteins were purified with affinity resins specific for NC-p65 and analyzed for enzyme activity, a single specific band of reaction was observed on gelatin- saturated zymograms.

AB - The NC-p65 cDNA is the first protease sequence cloned and described for Neospora caninum. The full length cDNA was isolated by 5'- and 3'-rapid amplification of cDNA ends (RACE). NC-p65 was composed of 865 amino acids with a predicted signal sequence, a proposed pro-domain, and an internal region of conserved repeats. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) and had a predicted active site made up of the catalytic residues, Asp 253, His 309, and Ser 484. Antibodies to recombinant NC-p65 recognized multiple bands on Neospora lysate immunoblots, but most intensely stained a 65 kDa band. When N. caninum proteins were purified with affinity resins specific for NC-p65 and analyzed for enzyme activity, a single specific band of reaction was observed on gelatin- saturated zymograms.

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