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 Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3-->Glu, Lys9--> Glu, Arg26-->Asp and Arg124-->Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (approximately 5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.
Nardone
E
E
Dibit, Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy.
Rosano
C
C
Santambrogio
P
P
Curnis
F
F
Corti
A
A
Magni
F
F
Siccardi
A G
AG
Paganelli
G
G
Losso
R
R
Apreda
B
B
Bolognesi
M
M
Sidoli
A
A
Arosio
P
P
D000818:Animals; D000906:Antibodies / Q000097:blood; D000941:Antigens / Q000276:immunology; D001360:Avidin / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D001667:Binding, Competitive; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002645:Chickens; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D007526:Isoelectric Point; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000276:immunology; D019809:Streptavidin / Q000032:analysis
D000906:Antibodies; D000941:Antigens; D011994:Recombinant Proteins; D001360:Avidin; D001710:Biotin; D019809:Streptavidin