ZFIN ID: ZDB-PUB-020403-20
Expression, purification and DNA-binding activity of tilapia muscle-specific transcription factor, MyoD, produced in Escherichia coli
Chen, Y.-H., Liang, C.-T., and Tsai, H.-J.
Date: 2002
Source: Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology   131(4): 795-805 (Journal)
Registered Authors: Chen, Yau-Hung, Tsai, Huai-Jen
Keywords: none
MeSH Terms:
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • DNA, Complementary/metabolism
  • Escherichia coli/metabolism*
  • Fishes
  • Genetic Vectors
  • Molecular Sequence Data
  • Muscle, Skeletal/metabolism
  • MyoD Protein/chemistry*
  • MyoD Protein/isolation & purification*
  • MyoD Protein/metabolism*
  • Peptides/chemistry
  • Phylogeny
  • RNA/metabolism
  • Recombinant Fusion Proteins/metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Species Specificity
  • Tilapia
PubMed: 11923092 Full text @ Comp. Biochem. Physiol. B Biochem. Mol. Biol.
MyoD is one of several helix-loop-helix proteins regulating muscle-specific gene expression. Using a reverse transcription-polymerase chain reaction, 5'-rapid cDNA end amplification, and plaque hybridization, MyoD cDNA was cloned from the mRNA of tilapia dorsal skeletal muscle. The 1015 bp MyoD cDNA product contained an 846 bp open reading frame with flanking regions of 115 and 64 bp at the 5'- and 3'-ends, respectively. Results showed that the tilapia MyoD sequence, which includes one polypeptide of 281 amino acids, shared sequence identities of 64.3, 64.1, 62.6 and 62.4% with those of zebrafish, carp, and two rainbow trout, respectively. Results from a molecular phylogenic tree assay showed that the tilapia MyoD was more closely related to those of other fishes than of higher vertebrates. Using Escherichia coli, a pET expression system, and an Ni(2+)-NTA column, we purified approximately 35 kDa recombinant tilapia MyoD. Results from an electrophoretic mobility shift assay demonstrated that the purified E. coli-produced tilapia MyoD was capable of binding to the DNA fragment sequence CA(C/T)(C/A)TG.