Cdc-like kinase 1 (CLK1) is a dual-specificity kinase capable of autophosphorylation on tyrosine residues and Ser/Thr phosphorylation of its substrates. CLK1 belongs to the CLK kinase family that regulates alternative splicing through phosphorylation of serine-arginine rich (SR) proteins. Recent studies have demonstrated that CLK1 has an important role in the replication of influenza A and chikungunya viruses. Furthermore, CLK1 was found to be relevant for the replication of HIV-1 and the West Nile virus, making CLK1 an interesting cellular candidate for the development of a host-directed antiviral therapy that might be efficient for treatment of newly emerging viruses. We describe here our attempts and detailed procedures to obtain the recombinant kinase domain of CLK1 in suitable amounts for crystallization in complex with specific inhibitors. The key solution for the reproducibility of crystals resides in devising and refining expression and purification protocols leading to homogeneous protein. Co-expression of CLK1 with λ-phosphatase and careful purification has yielded crystals of CLK1 complexed with the KH-CB19 inhibitor that diffracted to 1.65 Å. These results paved the path to the screening of more structures of CLK1 complexed compounds, leading to further optimization of their inhibitory activity. Moreover, since kinases are desired targets in numerous pathologies, the approach we report here, the co-expression of kinases with λ-phosphatase, previously used in other kinases, can be adopted as a general protocol in numerous kinase targets for obtaining reproducible and homogenic non-phosphorylated (inactive) forms suitable for biochemical and structural studies thus facilitating the development of novel inhibitors.

Cdc样激酶1（CLK1）是一种双特异性激酶，能够在酪氨酸残基上进行自身磷酸化，并使其底物的Ser / Thr磷酸化。CLK1属于CLK激酶家族，可通过富含丝氨酸精氨酸（SR）蛋白质的磷酸化来调节可变剪接。最近的研究表明，CLK1在A型流感和基孔肯雅病毒的复制中具有重要作用。此外，发现CLK1与HIV-1和西尼罗河病毒的复制有关，这使CLK1成为开发针对宿主的抗病毒疗法的有趣细胞候选物，这种疗法可能有效治疗新兴病毒。我们在这里描述了我们的尝试和详细过程，以获取适量的CLK1重组激酶结构域，可与特定抑制剂复合结晶。晶体可再现性的关键解决方案在于设计和完善表达和纯化方案，以产生均质的蛋白质。CLK1与λ磷酸酶的共表达和精心纯化已产生与KH-CB19抑制剂络合的CLK1晶体，衍射至1.65Å。这些结果为筛选更多结构的CLK1复合化合物铺平了道路，从而进一步优化了其抑制活性。此外，由于激酶是多种病理学中的理想靶标，因此我们在此报告的方法是激酶与以前在其他激酶中使用的λ-磷酸酶的共表达，