Separating Phosphorylation States Of A Kinase Using Ion Exchange Chromatography

In this case study we present our ability to express, phosphorylate and separate the native (dephosphorylated form) and the active (dual phosphorylated form) of a kinase to produce batches at high yield with excellent purity.

Many serine/threonine kinases are involved in  the regulation of essential cellular processes such as proliferation, differentiation, and survival. Often activation of the kinase occurs through dual phosphorylation at threonine residues by an upstream kinase, inducing a conformational change that enables full enzymatic activity. The diphosphorylated form therefore represents the active state of the kinase.

Figure 1: X-ray structure of a typical kinase

Within the Protein and Structure department at Sygnature Discovery we have significant experience expressing and purifying multiple forms of various kinases to best suit the particular downstream application. Recently we have had an internal project to optimise our workflow to produce both the inactive unphosphorylated and active diphosphorylated form of a particular kinase. This was with and without various tags and also with and without a biotinylated AVI tag to support SPR studies and other assay formats with the kinase.

Summary of the various forms that have been successfully produced in our laboratories.

• His-Avi-Kinase (biotinylated)
• His-Avi-ppKinase (biotinylated)
• Avi-Kinase (biotinylated)
• Avi-ppKinase (biotinylated)
• His-Kinase
• His-ppKinase
• Kinase
• ppKinase

Our purification process incorporates an ion-exchange chromatography step that effectively separates different phosphorylation states (Figure 2a). Using this optimised method, we consistently achieve high yields and high purity (non-p) Kinase and (pp)Kinase batches (Figure 2b–c). . Following ion exchange, we make use of our mass spectrometry system to analyse each fraction. We can then accurately determine which fractions contain non-phosphorylated kinase or ppKinase and pool accordingly for the downstream application (Figure 2d-e).

Figure 2: Purification and analysis of non-phospho Kinase and ppKinase samples. (a) Ion-exchange chromatography (IEX) profile showing separation of  phosphorylation states of the kinase. The initial IEX  peak and second peak contain non-phospho or ppKinase respectively.(b–c) SDS-PAGE analysis of purified (b) non-phospho Kinase and (c) ppKinase obtained using the optimized purification workflow. 

(d – e) Mass spectrometry (MS) analysis of final purified samples corresponding to (d) non-phosphorylated kinase, (e) ppKinase confirming identity and phosphorylation status. The diphosphorylated form was peptide mapped (data not shown) to confirm phosphorylation of both the expected Thr residues.

Building on this capability, Sygnature Discovery offers tailored solutions for partners seeking high-quality Kinases and their variants for structural studies, assay development, or drug discovery programs. Our optimized expression and purification platform ensures reproducible yields and purity across multiple tag formats, including biotinylated and phosphorylated forms, enabling flexibility for diverse applications such as binding studies, structural characterization, and screening campaigns. By combining technical expertise with scalable production, we provide a reliable resource for accelerating kinase-focused research and supporting clients in advancing their therapeutic pipelines.

Should these or other services be of interest please don’t hesitate to contact us info@peakproteins.com