Nardone E, Rosano C, Santambrogio P, Curnis F, Corti A, Magni F, Siccardi A G, Paganelli G, Losso R, Apreda B, Bolognesi M, Sidoli A, Arosio P
Dibit, Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy.
Eur J Biochem. 1998 Sep 1;256(2):453-60. doi: 10.1046/j.1432-1327.1998.2560453.x.
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.
由合成cDNA编码的成熟母鸡抗生物素蛋白以不溶性形式在大肠杆菌中表达。溶解、复性和纯化后，每升细胞培养物可获得约20 mg的产量。ELISA分析表明，天然抗生物素蛋白和重组抗生物素蛋白之间的生物素结合没有明显差异。此外，还产生了一种酸性抗生物素蛋白突变体，该突变体对四个暴露的碱性残基进行了Lys3→Glu、Lys9→Glu、Arg26→Asp和Arg124→Leu的替换。该蛋白作为野生型抗生物素蛋白表达和复性，其生物素结合活性未改变。突变体的酸性pI（约5.5）和缺乏聚集性使得在非变性条件下对其自身以及与生物素、生物素化转铁蛋白或过氧化物酶形成的复合物进行电泳分析变得容易。对致敏个体血清的分析表明，抗生物素蛋白突变体的抗原性发生了改变。两种重组抗生物素蛋白均已结晶，并通过分子置换法解析了三维结构，分辨率达到0.22 nm。在不存在生物素和糖基化的情况下，这两种重组分子的三维结构与先前报道的天然母鸡抗生物素蛋白的三维结构完全可比。
