CaMBP-10的cDNA克隆和表达及钙调素结合活性分析

Cloning and Expression of a cDNA Encoding CaMBP-10 and CaM Binding Activity Analysis

采用RT PCR法 ,从中国大白菜中分离了编码CaMBP 1 0的cDNA克隆 .该cDNA全长 4 96bp ,编码 92个氨基酸 ,3′端含有 2 1 6bp的非编码区和poly A尾 .将此BP 1 0cDNA的成熟蛋白序列导入表达质粒pET1 5b并转化至大肠杆菌E .coliBL2 1 (DE3)condonplus RIL进行表达 .以免疫印迹和钙调素结合分析法对重组BP 1 0进行鉴定 ,证明其保持了与天然BP 1 0相同的钙调素结合活性 .氨基酸和核苷酸序列分析结果显示 ,它与植物转脂蛋白高度同源 ,特别是含有 8个保守半胱氨酸 .BP 1 0与转脂蛋白之间具极为相似的理化性质如分子量、等电点、热稳定性等 .据此认为 ,CaMBP 1 0是转脂蛋白家族的新成员 ,Ca2 +

Calmodulin binding protein 10 (CaMBP 10) is a novel plant endogenous calmodulin binding protein with important physiological functions. The cDNA clone encoding for this protein was isolated from Chinese cabbage by using RT PCR. The full length of this cDNA is 496 bp, coding for 92 amino acid residues flanked by a 216 bp untranslated region at the 3′ end. The cDNA was inserted into an expression vector pET15b. The recombinant plasmid was expressed in E.coli BL21 (DE3)condonplus RIL. Using immunoblot and calmodulin binding assay, it was confirmed that this clone encoded CaMBP 10. CaMBP 10 showed very high amino acid and nucleotide sequence homology with plant nonspecific lipid transfer proteins (nsLTPs). Especially it also has eight conserved cysteine residues. In addition, there are striking resemblances between CaMBP 10 and plant nsLTP in their biochemical and physical properties. The results suggest that CaMBP 10 is a novel member of plant nsLTPs family,the Ca 2+ /CaM messenger system is involved in the function of lipid transfer protein.