Project Background
A European biotechnology company was developing a CRBN-recruiting PROTAC targeting a therapeutically important receptor tyrosine kinase (RTK) implicated in non-small cell lung cancer signaling. The client required milligram quantities of the active, phosphorylated kinase domain for warhead screening, binding validation, and ternary complex characterization. However, their internal attempts to produce soluble, properly folded kinase domain in E. coli resulted predominantly in inclusion bodies, and refolding efforts yielded material with inconsistent activity that could not support reproducible screening results.
Technical Challenges
The kinase domain required specific phosphorylation at the activation loop for proper catalytic conformation — a modification that bacterial systems cannot natively provide. The client also needed the protein in both phosphorylated (active) and dephosphorylated (inactive) forms to differentiate ATP-competitive warheads from allosteric binders. Additionally, the protein showed a tendency to aggregate at concentrations above 0.5 mg/mL, limiting its utility for SPR and crystallization trials.
BOC Sciences Solutions
- Multi-System Expression Evaluation: We tested expression in E. coli, baculovirus-infected Sf9 cells, and transient HEK293 systems in parallel. After comparing soluble yield, phosphorylation status, and monodispersity by analytical SEC across all three platforms, the baculovirus system was selected for scale-up production.
- Co-Expression with Activating Partners: To achieve uniform activation-loop phosphorylation, we co-expressed the kinase domain with its upstream activating kinase in the baculovirus system, generating homogeneously phosphorylated protein without chemical or enzymatic treatment.
- Stability Optimization and Quality Control: A buffer screen with thermal shift assay identified conditions that maintained the kinase in a monodisperse state at concentrations up to 5 mg/mL. Quality was confirmed by intact mass spectrometry, phosphosite mapping, and activity measurement using a peptide substrate assay.
Project Outcomes
BOC Sciences delivered 28 mg of purified, homogeneously phosphorylated kinase domain at >95% purity with confirmed monodispersity and catalytic activity. The client used this material to screen a focused library of 240 potential warhead candidates by SPR, identifying 18 binders with KD values below 1 μM. The protein also supported successful co-crystallization trials that yielded a 2.3 Å structure of the kinase domain bound to the lead warhead, providing the structural basis for subsequent PROTAC linker attachment point selection. Three PROTAC candidates derived from this campaign achieved DC50 values below 100 nM in target cell lines.
Project Background
A US-based pharmaceutical research group was pursuing a VHL-recruiting PROTAC against a G protein-coupled receptor (GPCR) target involved in inflammatory signaling. The client needed purified, functionally folded receptor protein for warhead binding studies and wanted to understand whether the receptor's conformational dynamics — particularly the equilibrium between active and inactive states — could be exploited for PROTAC-mediated degradation selectivity.
Technical Challenges
GPCRs are integral membrane proteins with seven transmembrane helices, making them notoriously difficult to express and purify in functional form. The receptor had limited structural information available, no reported high-affinity small molecule ligands suitable for direct PROTAC conversion, and required stabilization in detergent micelles or nanodiscs without losing ligand-binding competence. The client had attempted expression in E. coli and yeast without obtaining usable material.
BOC Sciences Solutions
- Construct Engineering and Expression Screening: We designed and screened 15 receptor constructs with different N-terminal truncations, fusion partners, and thermostabilizing point mutations across baculovirus and mammalian expression systems to identify variants with improved expression and stability.
- Detergent and Nanodisc Reconstitution Comparison: The receptor was purified in multiple membrane-mimetic environments — including DDM/CHS detergent micelles, MSP1D1 nanodiscs, and styrene-maleic acid (SMA) copolymer particles — to compare ligand-binding activity and conformational homogeneity across conditions.
- Ligand Binding and Conformational Profiling: We used SPR to measure binding of known orthosteric and allosteric ligands across the different reconstitution formats, and applied protein-ligand structure analysis by cryo-EM to visualize the receptor in both inactive and active-like conformations.
Project Outcomes
Among the 15 constructs tested, a thermostabilized variant expressed in baculovirus and reconstituted into nanodiscs showed the highest ligand-binding activity and conformational homogeneity. SPR measurements confirmed that the receptor in nanodiscs retained saturable, high-affinity binding to its endogenous peptide ligand (KD = 12 nM), and cryo-EM analysis at 3.4 Å resolution revealed a well-defined orthosteric pocket suitable for small molecule warhead development. The client used this material to initiate a fragment-based screening campaign that identified three distinct chemical starting points for PROTAC warhead optimization — a result that had not been achievable with their previous in-house expression efforts.
Integrated Target-to-Degradation Expertise
