Biochemical and structural properties of zebrafish Capsulin produced by Escherichia coli.
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 Escherichia coli. By circular dichroism spectroscopy, Capsulin was shown to be 55% α-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.
Chou
Chi-Yuan
CY
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan. cychou@ym.edu.tw
Hsu
Chia-Hao
CH
Wang
Yun-Hsin
YH
Chang
Min-Yen
MY
Chen
Li-Chao
LC
Cheng
Shu-Chun
SC
Chen
Yau-Hung
YH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010496:Pericardium / Q000166:cytology; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D014157:Transcription Factors / Q000032:analysis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D000906:Antibodies; D018076:DNA, Complementary; D011994:Recombinant Proteins; C556397:Tcf21 protein, zebrafish; D014157:Transcription Factors; D029961:Zebrafish Proteins