PUBLICATION
Biochemical and structural properties of zebrafish Capsulin produced by Escherichia coli
- Authors
- Chou, C.Y., Hsu, C.H., Wang, Y.H., Chang, M.Y., Chen, L.C., Cheng, S.C., and Chen, Y.H.
- ID
- ZDB-PUB-100719-53
- Date
- 2011
- Source
- Protein Expression and Purification 75(1): 21-27 (Journal)
- Registered Authors
- Chen, Yau-Hung
- Keywords
- analytical ultracentrifugation, anti-Capsulin antibody, bHLH structure, circular dichroism, recombinant Capsulin, nuclear localization
- MeSH Terms
-
- Zebrafish/genetics*
- Gene Expression
- Pericardium/cytology
- Cloning, Molecular
- Humans
- DNA, Complementary/genetics
- Transcription Factors/analysis*
- Transcription Factors/chemistry
- Transcription Factors/genetics*
- Transcription Factors/isolation & purification
- Amino Acid Sequence
- Animals
- Molecular Sequence Data
- Antibodies/immunology
- Cell Nucleus/ultrastructure
- Recombinant Proteins/analysis
- Recombinant Proteins/chemistry
- Recombinant Proteins/genetics
- Recombinant Proteins/isolation & purification
- Protein Multimerization
- Cell Line
- Transfection
- Escherichia coli/genetics*
- Sequence Alignment
- Zebrafish Proteins/analysis*
- Zebrafish Proteins/chemistry
- Zebrafish Proteins/genetics*
- Zebrafish Proteins/isolation & purification
- Protein Conformation
- PubMed
- 20627128 Full text @ Protein Expr. Purif.
Citation
Chou, C.Y., Hsu, C.H., Wang, Y.H., Chang, M.Y., Chen, L.C., Cheng, S.C., and Chen, Y.H. (2011) Biochemical and structural properties of zebrafish Capsulin produced by Escherichia coli. Protein Expression and Purification. 75(1):21-27.
Abstract
Capsulin is one of the transcription factors involved in regulating cell differentiation but its biochemical properties and structural characteristics are still unclear. In the present study, we cloned capsulin from zebrafish, which produces large numbers of transparent embryos and has well-characterized developmental stages. By alignment, the deduced amino acid sequence of zebrafish Capsulin, which contains a putative bHLH motif, shares very high homology to that of other species with an 72-82% identity. Zebrafish Capsulin was also targeted to the nucleus of mammalian cells when overexpressed by transient transfection. In order to characterize the structural and biochemical properties of zebrafish Capsulin, a recombinant zebrafish Capsulin protein was expressed and purified in E. coli. By circular dichroism spectroscopy, Capsulin was shown to be 55% alpha-helical. The size distribution assay by analytical ultracentrifugation indicated that it existed as a monomer-dimer mixture. The results suggested that the recombinant Capsulin has a well-organized and functional structure. Finally, endogenous Capsulin was distributed mainly in the epicardial cells of zebrafish by immunohistochemistry analysis using antibodies raised against zebrafish Capsulin. The present study not only helps us to comparatively analyze capsulin genes across species, but it also provides valuable structural information for further studies of Capsulin biological function in the future.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping