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.