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1.
Protein Expr Purif ; 182: 105842, 2021 06.
Article in English | MEDLINE | ID: mdl-33582289

ABSTRACT

Biologics are making up an increasing proportion of the global drug discovery pipeline. Supporting the expansion of biologics drug discovery requires higher throughput techniques for the expression, purification and characterization of both therapeutic candidates and reagents. Here we describe the programming and development of a novel ÄKTA™ instrument configuration that enables automated parallel and multistep chromatography over a range of scales. The programming strategy is offered as open source and the custom plumbing configuration was developed with off the shelf components available from Cytiva. Combined with high flow resin technology we show how this strategy can reduce the duration of a standard antibody purification process by 4.5X, from 4.5 h down to 1 h per run. An automated loading strategy was also developed to enable true walk away application of up to 24 samples and around the clock processing capability. The techniques used here to accomplish parallel multistep chromatography can be duplicated or modified for specific applications and represent a straightforward and cost-effective means to eliminate protein purification bottlenecks.


Subject(s)
Antibodies/isolation & purification , Automation, Laboratory , Chromatography, Affinity/instrumentation , Chromatography, Affinity/methods
2.
Mol Pharmacol ; 92(3): 310-317, 2017 09.
Article in English | MEDLINE | ID: mdl-28645932

ABSTRACT

The NaV1.7 voltage-gated sodium channel is implicated in human pain perception by genetics. Rare gain of function mutations in NaV1.7 lead to spontaneous pain in humans whereas loss of function mutations results in congenital insensitivity to pain. Hence, agents that specifically modulate the function of NaV1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high NaV1.7 expression have led to a surrogate target strategy approach employing chimeras with the bacterial channel NaVAb. In this report we describe the design, synthesis, purification, and characterization of a chimera containing part of the voltage sensor domain 2 (VSD2) of NaV1.7. Importantly, this chimera, DII S1-S4, forms functional sodium channels and is potently inhibited by the NaV1.7 VSD2 targeted peptide toxin ProTx-II. Further, we show by [125I]ProTx-II binding and surface plasmon resonance that the purified DII S1-S4 protein retains high affinity ProTx-II binding in detergent. We employed the purified DII S1-S4 protein to create a scintillation proximity assay suitable for high-throughput screening. The creation of a NaV1.7-NaVAb chimera with the VSD2 toxin binding site provides an important tool for the identification of novel NaV1.7 inhibitors and for structural studies to understand the toxin-channel interaction.


Subject(s)
Bacterial Proteins/chemistry , NAV1.7 Voltage-Gated Sodium Channel/physiology , Recombinant Fusion Proteins/chemistry , Spider Venoms/metabolism , Voltage-Gated Sodium Channels/chemistry , Bacterial Proteins/physiology , Binding Sites , HEK293 Cells , Humans , Surface Plasmon Resonance , Voltage-Gated Sodium Channels/physiology
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