BIOCHEMICAL-CHARACTERIZATION AND CRYSTAL-STRUCTURE OF A RECOMBINANT HEN AVIDIN AND ITS ACIDIC MUTANT EXPRESSED IN ESCHERICHIA-COLI

The mature hen avidin encoded by a synthetic cDNA was expressed in Esc herichia coli in an insoluble form. After resolubilization, renaturati on and purification, a recovery of about 20 mg/l cell culture was obta ined. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic a vidin mutant, bearing the substitutions Lys3-->Glu, Lys9-->Glu, Arg26- ->Asp and Arg124-->Leu of four exposed basic residues, was produced. T he protein, expressed and renatured as wild-type avidin, showed unalte red biotin-binding activity. The acidic pi (approximate to 5.5) and la ck of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its comple xes with biotin, biotinylated transferrin or peroxidase. Analysis of t he sera from sensitized subjects revealed that the avidin mutant has a ltered antigenicity. Both recombinant avidins were crystallized and th e three-dimensional structures solved by molecular replacement and re fined to 0.22 nm resolution. The three-dimensional structures of the t wo recombinant molecules, in the absence of biotin and of glycosylatio n, are fully comparable with those of the natural hen avidin previousl y reported.