Molecular cloning and expression of grass carp MyoD in yeast Pichia pastoris.
MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84(th) amino acids) and HLH domain (98-142(th) amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZalphaA and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.
Wang
Lixin
L
Pearl River Fisheries Research Institute, CAFS. Key Lab. of Tropical & Subtropical Fish Breeding & Cultivation of CAFS, Guangzhou 510380, P. R. China.
Bai
Junjie
J
Luo
Jianren
J
Chen
Hong
H
Ye
Xing
X
Jian
Qing
Q
Lao
Haihua
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics*; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D017570:MyoD Protein / Q000235:genetics* / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D017570:MyoD Protein; D011994:Recombinant Proteins