Custom RMP & IKKβ Proteins Reveal a New Sepsis Therapeutic Target | AtaGenix

Study Context

Sepsis kills over 14 million people annually. At its core, sepsis involves a runaway inflammatory cascade driven by NF-κB signaling in macrophages. While the pathway from TLR4 activation to IκB kinase β (IKKβ) phosphorylation is well characterized, the endogenous "brakes" on this system remain poorly understood. Identifying these negative regulators could open new therapeutic avenues beyond conventional immunosuppression.

A 2025 study by Shu-jie Pang and colleagues at Naval Medical University, published in Cell Communication and Signaling (DOI: 10.1186/s12964-025-02278-w), identified RNA polymerase II subunit 5-mediating protein (RMP) as one such brake. RMP binds directly to IKKβ and recruits protein phosphatase 2A (PP2A), dephosphorylating IKKβ and dampening NF-κB-driven inflammation. Crucially, a single point mutation (S439A) at RMP's phosphorylation site enhanced its binding to IKKβ — a finding that required carefully matched recombinant proteins to demonstrate convincingly.

Figure 1. Proposed mechanism: RMP binds IKKβ and recruits PP2A to dephosphorylate IKKβ, suppressing TLR4-induced NF-κB activation in macrophages during sepsis. The S439A mutation at RMP's phosphorylation site enhances IKKβ binding affinity.

The Challenge

Proving that RMP directly binds IKKβ — and that a single amino acid change at position 439 strengthens this interaction — required precisely matched recombinant proteins. The team needed four constructs in parallel: GST alone (negative control), GST-mouse RMP wild-type, GST-mouse RMP-S439A (phospho-dead mutant), and His-mouse IKKβ. All four had to be purified under comparable conditions so that any observed differences in binding (SPR) or kinase inhibition (ADP-Glo) could be attributed to biology, not production artifacts.

AtaGenix's Approach

AtaGenix designed a parallel E. coli expression and purification pipeline for all four proteins:

• Expression System: E. coli was the clear choice — RMP and IKKβ are intracellular signaling proteins that do not require glycosylation. The speed and yield of prokaryotic expression allowed all four constructs to be produced in a single batch cycle.
• Tag Strategy: GST tags for the three RMP variants (enabling glutathione-based pulldown assays) and a His tag for IKKβ (enabling orthogonal Ni-NTA purification and SPR chip immobilization). This dual-tag design allowed clean separation of bait and prey in interaction assays.
• Site-Directed Mutagenesis: The S439A mutation was introduced by AtaGenix's molecular biology team with full sequence verification, ensuring the mutant differed from wild-type by exactly one codon.
• Parallel Purification: All four proteins were purified under identical buffer and chromatography conditions (GST: glutathione agarose; His: Ni-NTA), eliminating purification-related variability from downstream comparisons.
• QC: SDS-PAGE and Western Blot confirmed identity, purity, and intact tags for all constructs before shipment.

Results and Impact

Using AtaGenix-produced proteins, the team generated three key findings:

• Direct binding confirmed by SPR: Real-time SPR (OpenSPR) demonstrated that RMP-WT binds IKKβ with measurable affinity, and the S439A mutant showed enhanced binding — providing the first biophysical evidence for this interaction.
• GST-pulldown validated the interaction: GST-RMP-WT and GST-RMP-S439A both pulled down His-IKKβ, with the mutant showing stronger co-purification. GST alone showed no binding, confirming specificity.
• Kinase inhibition quantified: ADP-Glo assays showed that RMP suppresses IKKβ kinase activity, consistent with its proposed role as a negative regulator of NF-κB signaling.

Figure 2. Surface plasmon resonance (SPR) analysis of RMP–IKKβ binding. Real-time sensorgrams demonstrate direct interaction between AtaGenix-produced His-IKKβ and GST-RMP proteins, with the S439A phospho-dead mutant showing enhanced binding compared to wild-type RMP.

Figure 3. GST-pulldown assay. GST-RMP-WT and GST-RMP-S439A both co-purified with His-IKKβ, while GST alone did not, confirming the specificity of the RMP–IKKβ interaction. The S439A mutant showed stronger binding, consistent with SPR data.

This case study is based on a published research collaboration. Results may vary depending on target protein, construct design, and project scope. All proprietary client information is subject to NDA.