Cloning and high-level expression of chicken apocytochrome c gene in Escherichia coli.
Chicken apocytochrome c gene with correct reading frame was easily cloned through excision by polymerase chain reaction of the intron in the genomic clone of chicken cytochrome c gene, and was successfully overexpressed in Escherichia coli by cloning into expression vector pET-3d under the control of T7 promoter. Expressed protein can amount to as high as 40% of the total protein and mainly presents as inclusion body. Purification of chicken apocytochrome c from the inclusion body and characterization by SDS-PAGE, isoelectric focusing electrophoresis, and amino acid analysis showed that the purified apocytochrome c is identical to that prepared from chicken heart cytochrome c by chemically depletion of heme.
National Laboratory of Biomacromolecules, Laboratory of Protein Engineering, Institute of Biophysics, Academia Sinica, Beijing, China.
J C
JC
Tong
L Q
LQ
Zhu
F Y
FY
Yang
D000818:Animals; D001059:Apoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D045304:Cytochromes c; D004926:Escherichia coli; D007438:Introns; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016364:Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping
D001059:Apoproteins; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D045304:Cytochromes c