胡萝卜抗冻蛋白的快速大量突变及原核表达

The Fast Mutation and Prokaryotic Expression of Carrot Antifreeze Protein

胡萝卜抗冻(DcAFP)具有典型的LRR特征,由约24个氨基酸残基组成重复单元,其中的保守基序是:P×××××L××L××L×LS×N×L×G×I,其中的保守天冬酰胺在与冰晶表面结合中起着关键的作用．为了探明该关键氨基酸对DcAFP重要性,需要对其进行一系列突变,以检测这些突变体的热滞可能发生的变化,而这需要构建大量的突变体．基于此,采用定点突变技术对该基序中保守的天冬酰胺进行了一系列的疏水性缬氨酸突变和亲水性的谷氨酰胺突变,并将这一系列突变体克隆至原核表达载体pET-11a中,在大肠杆菌BL21(DE3)中进行了高效表达与初步纯化,为后续测定其热滞活性的变化和验证天冬酰胺的重要性打下了基础．

The carrot antifreeze protein （DcAFP） possesses a typical leucine-rich feature （LRR）, containing tandem motifs of 24 amino acids （P×××××L××L××L×LS×N×L×G×I）, among which conservative asparagines play a key role in binding to ice surface. In order to ascertain the significance of these key asparagines, it is necessary to mutate asparagines serially and determine the possible change of these mutants. Therefore, the method of site-specific mutagenesis was used to turn conservative asparagines into hydrophobic valine or hydrophilic glutamine respectively. The serial mutants were inserted into prokaryotic expression vector pET-11a, and then were performed with high efficient expression and primary purification after transformed into E. coli BL21 （DE3）, providing theoretical bases for measuring the antifreeze activity change of DcAFP serial mutants and validating the significance of conservative asparagines.