Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii.
A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.
Pszenny
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Departamento de Parasitología Sanitaria, Instituto Nacional de Parasitología 'Dr M. Fatala Chaben', Av. Vélez Sarsfield 563 (1281), Buenos Aires, Argentina. vseni@ciudad.com.ar
Angel
S O
SO
Duschak
V G
VG
Paulino
M
M
Ledesma
B
B
Yabo
M I
MI
Guarnera
E
E
Ruiz
A M
AM
Bontempi
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D016054:DNA, Protozoan / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014122:Toxoplasma / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014359:Trypsin Inhibitor, Kazal Pancreatic
D018076:DNA, Complementary; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C412510:TgPI protein, Toxoplasma gondii; D014359:Trypsin Inhibitor, Kazal Pancreatic