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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 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.

SUBMITTER: Naik S

PROVIDER: S-EPMC4287005 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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

Naik Subhashchandra S Kumru Ozan S OS Cullom Melissa M Telikepalli Srivalli N SN Lindboe Elizabeth E Roop Taylor L TL Joshi Sangeeta B SB Amin Divya D Gao Phillip P Middaugh C Russell CR Volkin David B DB Fisher Mark T MT

Protein science : a publication of the Protein Society 20140728 10

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) an ...[more]

PMID: 25043635

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Project description:Sodium glucose transporters (SGLTs) belong to the mammalian solute carrier family SLC5. This family includes 12 different members in human that mediate the transport of sugars, vitamins, amino acids, or smaller organic ions such as choline. The SLC5 family belongs to the sodium symporter family (SSS), which encompasses transporters from all kingdoms of life. It furthermore shares similarity to the structural fold of the APC (amino acid-polyamine-organocation) transporter family. Three decades after the first molecular identification of the intestinal Na+-glucose cotransporter SGLT1 by expression cloning, many new discoveries have evolved, from mechanistic analysis to molecular genetics, structural biology, drug discovery, and clinical applications. All of these advances have greatly influenced physiology and medicine. While SGLT1 is essential for fast absorption of glucose and galactose in the intestine, the expression of SGLT2 is largely confined to the early part of the kidney proximal tubules, where it reabsorbs the bulk part of filtered glucose. SGLT2 has been successfully exploited by the pharmaceutical industry to develop effective new drugs for the treatment of diabetic patients. These SGLT2 inhibitors, termed gliflozins, also exhibit favorable nephroprotective effects and likely also cardioprotective effects. In addition, given the recent finding that SGLT2 is also expressed in tumors of pancreas and prostate and in glioblastoma, this opens the door to potential new therapeutic strategies for cancer treatment by specifically targeting SGLT2. Likewise, further discoveries related to the functional association of other SGLTs of the SLC5 family to human pathologies will open the door to potential new therapeutic strategies. We furthermore hope that the herein summarized information about the physiological roles of SGLTs and the therapeutic benefits of the gliflozins will be useful for our readers to better understand the molecular basis of the beneficial effects of these inhibitors, also in the context of the tubuloglomerular feedback (TGF), and the renin-angiotensin system (RAS). The detailed mechanisms underlying the clinical benefits of SGLT2 inhibition by gliflozins still warrant further investigation that may serve as a basis for future drug development.

Dataset Information

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

Publications

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

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