视黄醇结合蛋白L35和Q98位点突变及其对与视黄醇结合的影响

根据视黄醇结合蛋白 (RBP)晶体结构和计算机模拟对拟突变位点所作出的结构预测 ,筛选出 2个可能对RBP结合能力有影响的氨基酸位点 .利用定点突变技术分别将 35位亮氨酸突变为能引入较大构象变化的甘氨酸 ,将 98位谷氨酰胺突变为亲水、带正电的精氨酸 .将突变后的RBP基因转入表达宿主菌获得表达后 ,对突变体蛋白实现了变性条件下的分离纯化 .复性后与视黄醇的荧光增强饱和滴定试验表明 ,35位突变为甘氨酸后对RBP结合能力没有产生明显的影响 ,但 98位突变为精氨酸后 ,则显著的提高了与视黄醇的结合能力 ,说明 98位是影响RBP与视黄醇结合和解离的重要位点 .

Mutagenesis of L35 and Q98 on Human Retinol-binding Protein and Effects on Binding with Retinol

Retinol-binding protein(RBP)is a retinol-specific binding protein.To satisfy the need for cell differentiation,vision maintenance, reproduction and individual maturity, retinol is transported to target cells in a highly specific and regulated manner by combination with RBP and transthyretin.To determine amino acid residues in RBP which might be important for the binding of all trans-retinol,comparative model-building studies were performed.Based on three-dimensional X-ray crystallagraphic structure analysis, two specific amino acids were predicted to be important in retinol binding. The site-directed mutagenesis was performed to alter the L35 to G35, Q98 to R98 respectively. After expression and purification of mutant protein, flurescene binding assay was performed.L35G was found to have no apparent influence on the affinity for all trans-retinol,but the affinity of Q98R had been increased dramatically.