小鼠cAMP依赖的蛋白激酶催化亚基α的重组表达研究

Study on Expression of the Recombinant Mouse Catalytic Subunit α of cAMP-dependent Protein Kinase

将小鼠ｃＡＭＰ依赖的蛋白激酶（ｃＡＰＫ）催化亚基α（ｍＣα）分别以成熟、麦芽糖结合蛋白（ＭＢＰ）融合以及Ｎ端连续六个组氨酸（Ｈｉｓ_６）融合的形式在大肠杆菌中得到了高效表达，且成熟及融合的重组ｍＣα均有明显的蛋白激酶活性，表明蛋白激酶催化核心结构具有相对的独立性。其中Ｈｉｓ_６－ｍＣα可利用金属离子（Ｎｉ￣（２＋））配体亲和层析（Ⅰ－ＭＡＣ）一步纯化，所得融合蛋白可通过Ｈｉｓ_６亲合手臂（Ｔａｇ）固相化于金属离子（Ｎｉ￣（２＋））配体亲和树脂上，为进一步利用ＰｈａｇｅＤｉｓｐｌａｙ多肽库筛选ｃＡＰＫ识别的底物序列和专一性抑制剂打下了基础。

The catalytic subunit of mouse cAMP-dependent protein kinase (mCαwas highly expressed in E. coli as mature protein or MBP(Maltose Binding Protein)fusion protein or N terminal His_6 (six consecutive histidine residues) fusion protein. Since the recombinant mCα with orwithout fusion tags had obvious kinase activity, it inferred that the structure of the catalytic subunit of cAPK should be independent and intact. His_6 fusion mCα(His_6-mCα) was puified to near homogeneity by Immobilized Metal Chelation (Ni￣(2+)) Affinity Chromatography (IMAC) in one step. His_6-mCαcan be immobilized on Ni￣(2+)-IDA through His_6 affinity tags and used as ligands to facilitate the study of substrate specificity and specific inhibitors of cAPK by using phage display peptide libraries.