2cam: Difference between revisions

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.

2CAM is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Gallus_gallus Gallus gallus]. Structure known Active Site: . Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2CAM OCA].  

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