PUBLICATION
RETRACTED: Zebrafish caspase-3: molecular cloning, characterization, crystallization and phylogenetic analysis
- Authors
- Chakraborty, C., Nandi, S.S., Sinha, S., and Gera, V.K.
- ID
- ZDB-PUB-060724-10
- Date
- 2006
- Source
- Protein and Peptide Letters 13(6): 633-640 (Journal)
- Registered Authors
- Sinha, Surajit
- Keywords
- Zebrafish caspase-3, cloning, crystallization, phylogenetic analysis
- MeSH Terms
-
- Amino Acid Sequence
- Animals
- Caspase 3
- Caspases/chemistry*
- Caspases/genetics*
- Cell Line
- Cloning, Molecular
- Crystallization
- Crystallography, X-Ray
- DNA, Complementary
- Kidney/cytology
- Molecular Sequence Data
- Phylogeny*
- Recombinant Proteins/chemistry
- Recombinant Proteins/genetics
- Zebrafish*/genetics
- PubMed
- 16842121 Full text @ Protein Pept. Lett.
Citation
Chakraborty, C., Nandi, S.S., Sinha, S., and Gera, V.K. (2006) RETRACTED: Zebrafish caspase-3: molecular cloning, characterization, crystallization and phylogenetic analysis. Protein and Peptide Letters. 13(6):633-640.
Abstract
Apoptosis plays an important role in maintaining the normal function of various tissues and organs in different species. Caspase-3 is a terminal caspases which plays an important role in the execution of apoptosis in all vertebrates. It was cloned from zebra fish embryos and its properties were identified through Western blotting and biological activity. In the cells over-expressing caspase-3, Western blotting with an anti-His-tag antibody confirmed the presence of caspase-3 in the three bands that were proposed to correspond to the precursor form (33 kDa), the mature forms processed at the prodomain alone (29 kDa, large subunit) and small sub unit (13 kD). Fish kidney cells were transiently co-transfected with the beta-galactosidase reporter gene and either vector alone (mock), pZCASP3His (caspase-3) or pZCASP3His mutant (caspase-3 mutant). After 72 h following transfection of fish kidney cells, 35% of cells transfected with the zebra fish caspase-3 construct, pZCASP3His, showed apoptotic morphology when compared with cells transfected with the mock vector or an expression construct (pZCASP3His mutant) encoding the caspase-3 mutant lacking Cys. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. A thrombin cleavage recognition site was positioned at the fusion junction to release the caspase-3 from the fusion protein. Phylogenetic analysis showed that the cloned zebra fish caspase was a member of the caspase-3 subfamily with approximately 60% identity with caspase-3 from Xenopus, chicken and mammals. We have obtained structural information by X-ray crystallography. Orthorhombic crystals of the caspase-3 that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0 -7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 36.07 A, b = 38.80 A, c = 135.20 A.
Errata / Notes
This article is retracted by ZDB-PUB-220906-4.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping