Shrestha Amit, Hamilton Garth, O'Neill Eric, Knapp Stefan, Elkins Jonathan M
Structural Genomics Consortium, Oxford University, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
Gray Institute for Radiation Oncology and Biology, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
Protein Expr Purif. 2012 Jan;81(1):136-143. doi: 10.1016/j.pep.2011.09.012. Epub 2011 Oct 1.
Bacterial over-expression of kinases is often associated with high levels of auto-phosphorylation resulting in heterogeneous recombinant protein preparations or sometimes in insoluble protein. Here we present expression systems for nine kinases in Escherichia coli and, for the most heavily phosphorylated, the characterisation of factors affecting auto-phosphorylation. Experiments showed that the level of auto-phosphorylation was proportional to the rate of expression. Comparison of phosphorylation states following in vitro phosphorylation with phosphorylation states following expression in E. coli showed that the non-physiological 'hyper-phosphorylation' was occurring at sites that would require local unfolding to be accessible to a kinase active site. In contrast, auto-phosphorylation on unphosphorylated kinases that had been expressed in bacteria overexpressing λ-phosphatase was only observed on distinct exposed sites. Remarkably, the Ser/Thr kinase PLK4 auto-phosphorylated on a tyrosine residue (Tyr177) located in the activation segment. The results give support to a mechanism in which auto-phosphorylation occurs before or during protein folding. In addition, the expression systems and protocols presented will be a valuable resource to the research community.
激酶在细菌中的过表达通常与高水平的自磷酸化相关，这会导致重组蛋白制备物不均一，有时还会产生不溶性蛋白。在此，我们展示了大肠杆菌中九种激酶的表达系统，并针对磷酸化程度最高的激酶，对影响自磷酸化的因素进行了表征。实验表明，自磷酸化水平与表达速率成正比。体外磷酸化后的磷酸化状态与在大肠杆菌中表达后的磷酸化状态比较显示，非生理性的“过度磷酸化”发生在需要局部解折叠才能被激酶活性位点识别的位点。相反，在过表达λ-磷酸酶的细菌中表达的未磷酸化激酶上的自磷酸化仅在不同的暴露位点观察到。值得注意的是，丝氨酸/苏氨酸激酶PLK4在位于激活区的酪氨酸残基（Tyr177）上发生自磷酸化。这些结果支持了一种自磷酸化在蛋白质折叠之前或期间发生的机制。此外，所展示的表达系统和方案将为研究界提供宝贵的资源。
