Cloning and functional expression of a Boophilus microplus cathepsin L-like enzyme.
A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.
Renard
G
G
Centro de Biotecnologia do Estado do Rio Grande do Sul - UFRGS, C.P. 15005 - Campus do Vale, 91501-970 RS, Porto Alegre, Brazil.
Garcia
J F
JF
Cardoso
F C
FC
Richter
M F
MF
Sakanari
J A
JA
Ozaki
L S
LS
Termignoni
C
C
Masuda
A
A
D000818:Animals; D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013987:Ticks / Q000235:genetics*
D018076:DNA, Complementary; D004792:Enzyme Precursors; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L