Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio‐layer interferometry

Abstract

The ability of a GroEL-based bio-layer interferometry (BLI) assay to detect structurally altered and/or aggregated species of pharmaceutically relevant proteins is demonstrated. Assay development included optimizing biotinylated-GroEL immobilization to streptavidin biosensors, combined with biophysical and activity measurements showing native and biotinylated GroEL are both stable and active. First, acidic fibroblast growth factor (FGF-1) was incubated under conditions known to promote (40 C) and inhibit (heparin addition) molten globule formation. Heat exposed (40 C) FGF-1 exhibited binding to GroEL-biosensors, which was significantly diminished in the presence of heparin. Second, a polyclonal human IgG solution containing 6-8% non-native dimer showed an increase in higher molecular weight aggregates upon heating by size exclusion chromatography (SEC). The poly IgG solution displayed binding to GroEL-biosensors initially with progressively increased binding upon heating. Enriched preparations of the IgG dimers or monomers showed significant binding to GroEL-biosensors. Finally, a thermally treated IgG1 monoclonal antibody (mAb) solution also demonstrated increased GroEL-biosensor binding, but with different Additional Supporting Information may be found in the online version of this article. Subhashchandra Naik and Ozan S. Kumru contributed equally to the work. kinetics. The bound complexes could be partially to fully dissociated after ATP addition (i.e., specific GroEL binding) depending on the protein, environmental stress, and the assay's experimental conditions. Transmission electron microscopy (TEM) images of GroEL-mAb complexes, released from the biosensor, also confirmed interaction of bound complexes at the GroEL binding site with heat-stressed mAb. Results indicate that the GroEL-biosensor-BLI method can detect conformationally altered and/or early aggregation states of proteins, and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of therapeutic protein candidates.

Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs

“…Once assembled complexes are released from the small BLI biosensor tips into small volumes (e.g., 2–3 μl), the formation of large complexes (>100 kDa) can be easily verified using electron microscopy (Fig. 9; Naik et al 2013, 2014). …”

“…Furthermore, it is entirely possible that one can use EM validation of membrane protein insertion into lipid nanodiscs with small membrane proteins because insertion can be confirmed kinetically and by EM analysis. In the latter case, one could construct lipid nanodisc–small membrane protein complexes and validate the insertion of the protein into the nanodisc using specific monoclonal antibodies against the small membrane protein since antibody molecules are easily distinguished in an EM image field (Hernández-Rocamora et al 2012; Naik et al 2014). It is important to note that some commercially available MSPS that are used to form nanodiscs contain poly-histidine tags, enabling one with the ability to capture newly formed nanodiscs that can bind to Ni NTA BLI biosensor surfaces.…”

“…The advantage of using this micro-scale volume release approach is obvious since it can decrease testing times for optimal complex formation and can accelerate visualization of complex formation. This particular strategy of validating complexes using micro-scale samples for EM analysis from BLI platforms has also been used to evaluate and visualize protein–protein interactions between GroEL and pre-aggregates of therapeutic proteins (Naik et al 2014). …”

“…The ability to observe complex formation using label-free methods, removing the complex from these platforms, and depositing these directly onto electron microscopy grids will allow easy validation of assembly and insertion methods for scale up, eventually enabling one to rapidly generate pure samples to obtain high resolution structures (Naik et al 2013, 2014). By immobilizing and orientating soluble toxins prior to their insertion into nanodisc micelles, the crucial transition steps can be captured, allowing one to acquire high atomic resolution of toxin translocation competent states.…”

Assembly of cellulases with synthetic protein scaffolds in vitro

“…on a biosensor; changes in the reflected interference wave pattern between the sample and an internal reference layer result in a phase shift that can be followed in real time in both kinetic and quantitative modes [22]. All experiments were performed in kinetic buffer (20 mM PBS, pH 7.0; 1 mg/ml bovine serum albumin (BSA), and 0.02% Tween 20).…”