Cloning of a cysteine proteinase cDNA of adult Paragonimus westermani by polymerase chain reaction.
Cysteine proteinase cDNA fragment from adult mammalian trematode, Paragonimus westermani was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 804 bp. The deduced amino acid sequence consists of 268 amino acids. Sequence analysis and alignment showed significant sequence similarity to other eukaryotic cysteine proteinases and conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. The cysteine proteinase cDNA fragment was also subcloned in the expression vector pET and expressed as a C-terminal His-tag fusion protein in Escherichia coli.
Park
H
H
Department of Parasitology and Biochemistry, College of Medicine, Wonkwang University, Iksan, Korea.
Hong
K M
KM
Ryu
J S
JS
Shin
C H
CH
Lee
J B
JB
Soh
C T
CT
Paik
M K
MK
Min
D Y
DY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D018509:DNA, Helminth / Q000235:genetics*; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010238:Paragonimus / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D018509:DNA, Helminth; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases