Hemmer W, McGlone M, Taylor S S
Department of Chemistry and Biochemistry, University of California, San Diego 92093-0654, USA.
Anal Biochem. 1997 Feb 15;245(2):115-22. doi: 10.1006/abio.1996.9952.
Knowledge of the crystal structure of the catalytic subunit (C) of cAMP-dependent protein kinase provided for the first time a molecular basis for probing function by site-directed mutagenesis. The purification of mutant C-subunits, however, presented new and unanticipated challenges due to instability, insolubility, and underphosphorylation of the altered proteins. To overcome these barriers, a rapid and efficient method for purifying recombinantly expressed C-subunits was developed. Purification to near homogeneity is achieved in less than 5 h. The procedure is based on colysis of bacteria that overexpress the C-subunit with bacteria that overexpress a poly-His-tagged mutant of the type II regulatory subunit H6RII (R213K). This mutant R-subunit with an altered cAMP binding site A forms holoenzyme rapidly in bacterial extracts, and the Ka (cAMP) for the resulting holoenzyme, 27-37 microM, is nearly 50-fold increased compared to holoenzyme formed with wild-type RII. Thus, after batchwise immobilizing the holoenzyme on Ni(2+)-resin, the free C-subunit can be directly eluted batchwise with high concentrations of cAMP. The method is described for the purification of wild-type C, with yields of approximately 5 mg/liter. In addition, a mutant subunit, C[G52S], which is defective in ATP binding and could not be isolated using previously described methods, was purified with equal efficiency.
环磷酸腺苷（cAMP）依赖性蛋白激酶催化亚基（C）晶体结构的知识首次为通过定点诱变探究功能提供了分子基础。然而，由于突变C亚基的不稳定性、不溶性和磷酸化不足，其纯化带来了新的、意想不到的挑战。为克服这些障碍，开发了一种快速高效的纯化重组表达C亚基的方法。不到5小时即可实现纯化至近乎同质。该方法基于用过量表达II型调节亚基H6RII（R213K）的多组氨酸标签突变体的细菌裂解过量表达C亚基的细菌。这种cAMP结合位点A发生改变的突变R亚基在细菌提取物中迅速形成全酶，所得全酶的Ka（cAMP）为27 - 37 microM，与野生型RII形成的全酶相比增加了近50倍。因此，在将全酶分批固定在Ni(2+)树脂上后，游离的C亚基可以直接用高浓度的cAMP分批洗脱。该方法用于纯化野生型C，产量约为5 mg/升。此外，一种在ATP结合方面有缺陷且无法用先前描述的方法分离的突变亚基C[G52S]，也以相同效率得到了纯化。
