Cloning, expression, and structural analysis of recombinant BJcuL, a c-type lectin from the Bothrops jararacussu snake venom.
The lactose-binding lectin from Bothrops jararacussu venom (BJcuL) is a homodimer belonging to group VII of the c-type animal lectins. BJcuL has also been shown to serve as an interesting tool for combating tumor progression by inhibiting cancer and endothelial cell growth. However, detailed structural studies of BJcuL and its biological mechanisms of cytotoxicity are yet to be reported, perhaps because of the non-availability of recombinant proteins in necessary quantities. Intending to increase the present information about structural and consequently the understating of biological studies, the cDNA coding for BJcuL from a venom gland has been cloned and sequenced. The mature protein-coding region was amplified by PCR with specific oligonucleotides, and subcloned into the pET-15b vector to express the recombinant BJcuL in Escherichia coli BL21 (DE3). The deduced amino acid sequence exhibits a high degree of sequence identity with c-type lectins (CTLs) and c-type lectin-like domains (CTLDs). An insoluble and inactive 18.5-kDa protein was overexpressed after 1.0mM IPTG induction. The recombinant BJcuL was recovered and denatured in a buffer with 6M urea and purified on a nickel-affinity column. Protein refolding was carried out on this column, during procedure purification, followed by dialysis against CTBS and then by gel filtration for separation of the active dimmer. The refolding process of rBJcuL and the analysis of its structure were confirmed by biological assay, circular dichroism, and MALDI-TOF.
Kassab
Bayki H
BH
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109, Campinas-SP 13083-970, Brazil. kassab@unicamp.br
de Carvalho
Daniela D
DD
Oliveira
Marcos A
MA
Baptista
Gandhi R
GR
Pereira
Gonçalo A G
GA
Novello
José C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017837:Bothrops; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D037181:Lectins, C-Type / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
C498078:BJcuL lectin, Bothrops jararacussu; D003435:Crotalid Venoms; D018076:DNA, Complementary; D037181:Lectins, C-Type; D011994:Recombinant Proteins