Delivering Fit-for-Purpose Interleukin for Structural and Functional Studies

Advancing Interleukin Research Through Tailored Protein Production

Interleukins (ILs) are a family of cytokines that play an important role in immunology and are the focus of many Oncology research programs. However, to support their work researchers often require a purified IL protein construct with specific characteristics to support the desired end use, be it structural biology, assay development or functional studies. In this case study we present how Sygnature Discovery’s in‑house protein science team developed a parallel‑track production strategy using E. coli and mammalian HEK293 expression systems to produce an Interleukin (IL-X) for a client who needed the protein in different forms for both structure and assay development.

To generate structural-grade protein, Sygnature Discovery optimized a literature refolding and purification protocol from bacterial inclusion bodies. The result was a dramatic increase in final protein yield while producing homogeneous PTM-free protein suitable for crystallography and NMR studies. In parallel, we used HEK293 cells to produce fully functional glycosylated IL-X to support biochemical assay experiments.

This case study demonstrates Sygnature Discovery’s ability to develop tailored expression and purification strategies for challenging proteins, enabling the delivery of fit-for-purpose protein for downstream research applications.

Sygnature’s Approach & Solutions

Overcoming Inclusion Body Impurities

While mammalian expression and purification of IL-X was relatively straightforward via standard protocols, procuring IL-X from E. coli inclusion bodies required additional optimization. The first improvement to literature protocol focused on protein expression.  rather than relying on standard expression conditions, Sygnature Discovery extended the post-induction incubation and refined the temperature conditions.  

Removing Impurities that would Compromise Refolding

An initial evaluation of the isolated inclusion bodies (IB) revealed substantial impurities were still present despite a series of IB wash steps (Figure 1a). Following inclusion body (IB) solubilization, a denatured Immobilized Metal Affinity Chromatography (IMAC) step was introduced, with urea retained in the buffer, to remove the majority of impurities prior to refolding (Figure 1b). This enabled selective isolation of interleukin from host-cell contaminants and therefore a much purer starting point for the refolding of the IL-X.

Improving the Refolding Protocol

The IMAC purified, denatured interleukin underwent a tailored refolding process, based on our in-house knowledge of working with other interleukins and cytokines, generating a soluble fully folded form that was then suitable for further chromatographic capture and purification (Figure 2).

Optimizing Protein Yield on IEX

High sample conductivity initially prevented good interleukin capture via ion exchange chromatography (IEX). To overcome this incompatibility, we diluted the load sample with a low-salt buffer which then improved column binding and increased the IEX elution yield by 12-fold (Figure 3). 

As a final polishing step, the pool was subjected to size exclusion chromatography, isolating pure interleukin and completing the production pathway.

Results & Impact

What Sygnature Delivered

• Structural-grade PTM-free protein delivered for crystallography and NMR (Figure 4a).
• Glycosylated interleukin delivered for biochemical assays (Figure 4b).
• By pursuing a parallel-cloning and expression strategy we delivered interleukin formats tailored for their intended end use.

Together, these optimizations enabled the delivery of both structural-grade and assay-ready protein within a single coordinated program. This demonstrates Sygnature Discovery’s ability to tailor protein production strategies to diverse downstream applications.

Why Sygnature Discovery?

Parallel-Expression Strategy

• Bacterial and mammalian expression platforms run in parallel to deliver post-translational profiles tailored to downstream applications, ensuring both interleukin formats could be delivered within one program.

Specialized Expertise in Refolding Challenging Cytokines

• Delivered a refolding and purification protocol supporting an improved yield (over the literature protocol) of PTM‑free protein suitable for structural studies.

Cross-Functional Collaboration

• Protein Science at Sygnature Discovery means access to collaborative expertise striving to optimize yield, purity, and structural suitability for downstream applications.