Medley Q G, Lee S F, Côté G P
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Protein Expr Purif. 1990 Nov;1(2):155-8. doi: 10.1016/1046-5928(90)90010-v.
The initial step in the purification of Dictyostelium myosin II heavy chain kinase A (MHCK A) is chromatography over phosphocellulose. Fractions containing MHCK A are pooled and chromatographed over a Mono Q column (Pharmacia LKB Biotechnology) equilibrated in 0.15 M KCl. Under these conditions MHCK A and most of the contaminating proteins elute in the flowthrough. The addition of Mg2+ and ATP to the Mono Q flowthrough results in the phosphorylation, within 15 min, of MHCK A to a level of 10 mol of phosphate per mole of 130-kDa kinase subunit. The hyperphosphorylated MHCK A binds to Mono Q columns in the presence of 0.15 M KCl and can be eluted, as a single homogeneous band, by a salt gradient to 0.35 M KCl. A similar purification procedure may prove useful for other proteins which can be highly phosphorylated. Hyperphosphorylation is shown to have no effect on the position at which MHCK A elutes from gel filtration columns (apparent M(r) greater than 700,000).
盘基网柄菌肌球蛋白II重链激酶A（MHCK A）纯化的第一步是通过磷酸纤维素进行层析。含有MHCK A的组分合并后，在平衡于0.15 M KCl的Mono Q柱（Pharmacia LKB生物技术公司）上进行层析。在这些条件下，MHCK A和大多数污染蛋白在流穿液中洗脱。向Mono Q流穿液中加入Mg2+和ATP，15分钟内MHCK A被磷酸化至每摩尔130 kDa激酶亚基含10摩尔磷酸的水平。在0.15 M KCl存在下，过度磷酸化的MHCK A与Mono Q柱结合，并可通过盐梯度至0.35 M KCl作为单一均一的条带洗脱。类似的纯化程序可能对其他可被高度磷酸化的蛋白质有用。结果表明，过度磷酸化对MHCK A从凝胶过滤柱上洗脱的位置没有影响（表观分子量大于700,000）。
