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Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.

ABSTRACT: Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.

SUBMITTER: Zhou T

PROVIDER: S-EPMC7550166 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Publications

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.

Zhou Tongqing T Teng I-Ting IT Olia Adam S AS Cerutti Gabriele G Gorman Jason J Nazzari Alexandra A Shi Wei W Tsybovsky Yaroslav Y Wang Lingshu L Wang Shuishu S Zhang Baoshan B Zhang Yi Y Katsamba Phinikoula S PS Petrova Yuliya Y Banach Bailey B BB Fahad Ahmed S AS Liu Lihong L Lopez Acevedo Sheila N SN Madan Bharat B Oliveira de Souza Matheus M Pan Xiaoli X Wang Pengfei P Wolfe Jacy R JR Yin Michael M Ho David D DD Phung Emily E DiPiazza Anthony A Chang Lauren A LA Abiona Olubukola M OM Corbett Kizzmekia S KS DeKosky Brandon J BJ Graham Barney S BS Mascola John R JR Misasi John J Ruckwardt Tracy T Sullivan Nancy J NJ Shapiro Lawrence L Kwong Peter D PD

Cell reports 20201012 4

Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as wel ...[more]

PMID: 33091382

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Project description:Fractionation is essential to achieving deep proteome coverage for sample multiplexing experiments where currently up to 18 samples can be analyzed concurrently. However, peptide fractionation (i.e., upstream of LC-MS/MS analysis) with a liquid chromatography system constrains sample processing as only a single sample can be fractionated at once. Here, we highlight the use of spin column-based methods which permit multiple multiplexed samples to be fractionated simultaneously. These methods require only a centrifuge and eliminate the need for a dedicated liquid chromatography system. We investigate peptide fractionation with strong anion exchange (SAX) and high-pH reversed phase (HPRP) spin columns, as well as a combination of both. In two separate experiments, we acquired deep proteome coverage (>8000 quantified proteins), while starting with <25 μg of protein per channel. Our datasets showcase the proteome alterations in two human cell lines resulting from treatment with inhibitors acting on the ubiquitin-proteasome system. We recommend this spin column-based peptide fractionation strategy for high-throughput screening applications or whenever a liquid chromatograph is not readily available. SIGNIFICANCE: Fractionation is a means to achieve deep proteome coverage for global proteomics analysis. Typical liquid chromatography systems may be a prohibitive expense for many laboratories. Here, we investigate prefractionation with strong anion exchange (SAX) and high-pH reversed phase (HPRP) spin columns, as well as a combination of both, as peptide fractionation methods. These spin columns have advantages over liquid chromatography systems, which include relative affordability, higher throughput capability, no carry over, and fewer potential instrument-related malfunctions. In two separate experiments, we acquired deep proteome coverage (>8000 quantified proteins), thereby showing the utility of each or a combination of both spin columns for global proteome analysis.

Dataset Information

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.

Publications

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.

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