Expression, purification and DNA-binding activity of tilapia muscle-specific transcription factor, MyoD, produced in Escherichia coli.
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.
Chen
Yau Hung
YH
Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan, ROC.
Liang
Chin Tien
CT
Tsai
Huai Jen
HJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005399:Fishes; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D017570:MyoD Protein / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D017210:Tilapia
D018076:DNA, Complementary; D017570:MyoD Protein; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012313:RNA