Rajamohan Francis, Harris Melissa S, Frisbie Richard K, Hoth Lise R, Geoghegan Kieran F, Valentine James J, Reyes Allan R, Landro James A, Qiu Xiayang, Kurumbail Ravi G
Pfizer Global Research and Development, Groton, CT 06340, USA.
Protein Expr Purif. 2010 Oct;73(2):189-97. doi: 10.1016/j.pep.2010.04.022. Epub 2010 May 6.
AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine protein kinase that plays a central role in whole-body energy homeostasis. AMPK is a heterotrimeric enzyme with a catalytic (alpha) subunit and two regulatory (beta and gamma) subunits. The muscle-specific AMPK heterotrimeric complex (alpha2beta2gamma3) is involved in glucose and fat metabolism in skeletal muscle and therefore has emerged as an attractive target for drug development for diabetes and metabolic syndrome. To date, expression of recombinant full-length human AMPK alpha2beta2gamma3 has not been reported. Here we describe the expression, purification and biochemical characterization of functional full-length AMPK alpha2beta2gamma3 heterotrimeric complex using an Escherichia coli expression system. All three subunits of AMPK alpha2beta2gamma3 were transcribed as a single tricistronic transcript driven by the T7 RNA polymerase promoter, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. The self-assembled trimeric complex was purified from the cell lysate by nickel-ion chromatography using the hexahistidine tag fused exclusively at the N-terminus of the alpha 2 domain. The un-assembled beta 2 and gamma 3 domains were removed by extensive washing of the column. Further purification of the heterotrimer was performed using size exclusion chromatography. The final yield of the recombinant AMPK alpha2beta2gamma3 complex was 1.1mg/L culture in shaker flasks. The E. coli expressed enzyme was catalytically inactive after purification, but was activated in vitro by upstream kinases such as CaMKKbeta and LKB1. The kinase activity of activated AMPK alpha2beta2gamma3 complex was significantly enhanced by AMP (an allosteric activator) but not by thienopyridone A-769662, a known small molecule activator of AMPK. Mass spectrometric characterization of recombinant AMPK alpha2beta2gamma3 showed significant heterogeneity before and after activation that could potentially hamper crystallographic studies of this complex.
AMP激活的蛋白激酶（AMPK）是一种能量感应丝氨酸/苏氨酸蛋白激酶，在全身能量稳态中起核心作用。AMPK是一种异源三聚体酶，由一个催化（α）亚基和两个调节（β和γ）亚基组成。肌肉特异性AMPK异源三聚体复合物（α2β2γ3）参与骨骼肌中的葡萄糖和脂肪代谢，因此已成为糖尿病和代谢综合征药物开发的一个有吸引力的靶点。迄今为止，尚未报道重组全长人AMPKα2β2γ3的表达。在此，我们描述了使用大肠杆菌表达系统对功能性全长AMPKα2β2γ3异源三聚体复合物的表达、纯化及生化特性分析。AMPKα2β2γ3的所有三个亚基均作为由T7 RNA聚合酶启动子驱动的单个多顺反子转录本进行转录，从而允许在细菌胞质溶胶中自发形成异源三聚体复合物。通过使用仅融合在α2结构域N端的六聚组氨酸标签，通过镍离子色谱从细胞裂解物中纯化自组装三聚体复合物。通过对柱子进行大量洗涤，去除未组装的β2和γ3结构域。使用尺寸排阻色谱对异源三聚体进行进一步纯化。在摇瓶中，重组AMPKα2β2γ3复合物的最终产量为1.1mg/L培养物。纯化后，大肠杆菌表达的酶无催化活性，但在体外可被上游激酶如CaMKKβ和LKB1激活。变构激活剂AMP可显著增强激活的AMPKα2β2γ3复合物的激酶活性，但已知的AMPK小分子激活剂噻吩并吡啶A-769662则无此作用。重组AMPKα2β2γ3的质谱表征显示，激活前后存在显著的异质性，这可能会妨碍对该复合物的晶体学研究。
