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将小鼠ｃＡＭＰ依赖蛋白激酶催化亚基α（ｍＣα）基因从３′端截去９６ｂｐ后与Ｈｉｓ－ｔａｇ融合，构建Ｃ端融合Ｈｉｓ６的表达质粒ｐＺＰｍＣα４Ｈ。该质粒在大肠杆菌ＢＬ２１（ＤＥ３）中得到了高效表达，目的蛋白ｍＣα４Ｈ的表达量与ｍＣα相近。该蛋白在３７℃表达时主要以包含体形式存在，而２２℃表达时则大部分可溶。利用固定化金属离子（Ｎｉ２＋）配体亲和层析，分别从破菌上清液和包含体中纯化ｍＣα４Ｈ，结果表明通过一步亲和结合纯化，从破菌上清液中得到的ｍＣα４Ｈ蛋白纯度在７５％左右，而从包含体中得到的ｍＣα４Ｈ蛋白纯度可达９０％以上。纯化ｍＣα４Ｈ的体外和体内豆蔻酰化分析结果表明它具有与ｍＣα一致的ＮＭＴ底物活性。
The mouse cAMP-dependent protein kinase catalytic subunit α (mCα) gene was cloned from the 3 ’end of 96bp and then fused with His-tag to construct the C-terminal fusion His6 expression plasmid pZPmCα4H. The plasmid was highly expressed in E. coli BL21 (DE3), and the expression level of the target protein mCα4H was similar to that of mCα. The protein is mainly present in inclusion bodies when expressed at 37 ° C and mostly soluble at 22 ° C. The mCα4H protein was purified from the broken bacteria supernatant and inclusion body respectively by immobilized metal ion (Ni2 +) ligand affinity chromatography. The results showed that the mCα4H protein purity obtained from the broken bacteria supernatant by one-step affinity binding purification Around 75%, while the purity of mCα4H protein obtained from the inclusion body can reach more than 90%. In vitro and in vivo myristoylation analysis of purified mCalphaH shows that it has an NMT substrate activity that is consistent with mCa.