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

Project Snapshot — Naval Medical University researchers needed matched sets of GST-tagged RMP variants and His-tagged IKKβ to map a previously unknown protein-protein interaction in sepsis-driven inflammation. AtaGenix delivered four high-purity E. coli-expressed proteins — including a critical S439A point mutant — that enabled SPR, GST-pulldown, and kinase assays, culminating in a publication in Cell Communication and Signaling (2025).

Fig. 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 enhances IKKβ binding affinity. Adapted from Cell Commun Signal 2025.

Four Parallel Constructs: GST alone (negative control), GST-RMP wild-type, GST-RMP-S439A (phospho-dead mutant), and His-IKKβ — all had to be produced and purified under identical conditions.

Single-Residue Sensitivity: The S439A mutation produces subtle binding differences only detectable when production variables are eliminated.

Dual-Tag Strategy: GST tags for pulldown and His tag for orthogonal Ni-NTA purification and SPR chip immobilization required careful design.

Multi-Assay Compatibility: Proteins needed to perform in SPR, GST-pulldown, and kinase activity assays without buffer or purity artifacts.

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.

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.

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.

Fig. 2. SPR analysis of RMP–IKKβ binding. Real-time sensorgrams demonstrate direct interaction between His-IKKβ and GST-RMP proteins, with the S439A mutant showing enhanced binding. Adapted from Cell Commun Signal 2025.

Fig. 3. GST-pulldown assay. GST-RMP-WT and GST-RMP-S439A both co-purified with His-IKKβ, while GST alone did not. The S439A mutant showed stronger binding, consistent with SPR data. Adapted from Cell Commun Signal 2025.

Why This Matters

Protein-protein interaction studies that compare wild-type vs. point mutants demand exceptional consistency between reagents. A single-residue change like S439A can produce subtle binding differences that are only detectable when production variables are eliminated. This project illustrates how AtaGenix's parallel expression pipeline — same host, same conditions, same purification — gives researchers the confidence to attribute observed differences to biology, not batch variation.

Need matched protein sets for interaction studies, kinase assays, or structural biology? AtaGenix delivers parallel-produced wild-type and mutant proteins under identical conditions.

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