SITE-DIRECTED MUTAGENESIS AT GLU44 AND GL U56 OF CYTOCHROME B(5) AND THEIR STRUCTURAL COMPARISON WITH WILD-TYPE PROTEIN

The codon of Glu44 and Glu56, GAA in the gene of trypsin-solubilized b ovine liver microsomal cytochrome b(5) (82 residues in length) was cha nged into GCT coding for Ala by oligonucleotide site-directed mutagene sis and three cytochrome b(5) mutants of E44A, E56A, and E44/56A were obtained, The mutant genes were ligated into E(co)R I/Hind III-cut pUC 19 and the resulting plasmid was transformed into JM83. All of them we re expressed in E. coli successfully, The bacteria containing wild typ e or mutant cytochrome b(5) were dealt with lysozyme, deoxyribonucleas e and ribonuclease, then the proteins were isolated and purified by am monium sulfate precipitation, ion-exchange chromatography and gel filt ration, The UV-visible and circular dichroism spectra of purified prot eins were studied, The results show that the mutagenesis at the surfac e residues does not alter the structure of cytochrome b(5) significant ly.