Abstract: The codon of Glu44 and Glu56,GAAin the gene of trypsin-solubilized bovine liver microsomal cytochrome b₅(82 residues in length)was changed into GCTcoding for Ala by oligonucleotide site-directed mutagenesis and three cytochrome b₅ mutants of E44A,E56A,and E44/56 Awere obtained.The mutant genes were ligated into ECORⅠ/Hind Ⅲ-cut pUC19 and the resulting plasmid was transformed into JM83.All of them were expressed in E. coli successfully.The bacteria containing wild type or mutant cytochrome b₅ were dealt with lysozyme, deoxyribonuclease and ribonuclease,then the proteins were isolated and purified by ammonium sulfate precipitation,ion-exchange chromatography and gel filtration.The UV-visible and circular dichroism spectra of purified proteins were studied. The results show that the mutagenesis at the surface residues does not alter the structure of cytochrome b₅ significantly.

Key words: Cytochrome b₅, Site-directed mutagenesis, Protein structure