Generation of synthetic nanobodies against delicate proteins

Zimmermann, Iwan; Egloff, Pascal; Hutter, Cedric A. J.; Kuhn, Benedikt T.; Bräuer, Philipp; Newstead, Simon; Dawson, Roger; Geertsma, Eric R.; Seeger, Markus A.

doi:10.1038/s41596-020-0304-x

Cited by 134 publications

(177 citation statements)

References 58 publications

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“…The uniformity of amplification of these DNA library members is extremely important to avoid their loss. This is particularly relevant to quantitative analysis of metagenomes and amplification of DNA sequences selected via phage (25,26), ribosome (27), and messenger RNA (28) display. Although different approaches to DNA library amplification were proposed (23,29,30), alternative methodologies are still in demand.…”

Section: Discussionmentioning

confidence: 99%

Liquid drop of DNA libraries reveals total genome information

Terekhov

Eliseev

Ovchinnikova

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Conventional “bulk” PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

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Section: Discussionmentioning

confidence: 99%

Liquid drop of DNA libraries reveals total genome information

Terekhov

Eliseev

Ovchinnikova

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…The RBD was eluted using 300 mM of imidazole in Buffer A. For biotinylation 26,27 , the purified RBD with the Avi-tag intact (0.8 mg mL -1 ) was incubated with 5 mM ATP, 10 mM magnesium acetate, 43.5 μM biotin, 22 g mL -1 home-purified BirA in 3.2 mL volume and incubated at 4 °C for 16 h. Biotinylated RBD was concentrated using a 10-kDa cut-off membrane concentrator to ~3 mg mL -1 before loaded onto a Superdex Increase 200 10/300 GL column for size exclusion chromatography. Fractions containing the RBD were pooled, aliquoted, flash-frozen in liquid nitrogen, and stored at -80 °C before use.…”

Section: Protein Expression and Purification -Sars-cov-2 S-rbd For Symentioning

confidence: 99%

“…Sybodies were expressed with a C-terminally His-tag in Escherichia coli MC1061 cells. Briefly, cells carrying sybody genes in the vector pSb-init 26,27 were grown in…”

Section: Protein Expression and Purification -Sybodies In Escherichiamentioning

confidence: 99%

“…Sybody selection was performed using a combination of ribosome display and phage display 26,27 . In vitro translation of the 'Concave', 'Loop', and 'Convex' library was Binders can be identified based on earlier retention volume, presumably reflecting the bigger size of the FL-RBDavi-sybody complex than the FL-RBDavi alone.…”

Section: Sybody Selection -Ribosome Display and Phage Displaymentioning

confidence: 99%

“…Llama-derived nanobodies are generally more heat stable, easier and less expensive for production, and more amenable to protein engineering compared to conventional antibodies 24 . As single-chain antibodies, nanobody libraries are less complex to construct and screen, enabling in vitro selection of high-affinity binders in relative short time, typically 2-4 weeks [25][26][27][28][29][30] . Recently, several nanobody therapeutics, including the caplacizumab approved by the US Food and Drug Administration, have been developed for a variety of immune diseases 31 .…”

Section: Introductionmentioning

confidence: 99%

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A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

Cai

et al. 2020

Preprint

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SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

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From in vitro towards in situ: structure‐based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

et al. 2020

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ATP-binding cassette (ABC) exporters have been studied now for more than four decades, and recent structural investigation has produced a large number of protein database entries. Yet, important questions about how ABC exporters function at the molecular level remain debated, such as which are the molecular recognition hotspots and the allosteric couplings dynamically regulating the communication between the catalytic cycle and the export of substrates. This conundrum mainly arises from technical limitations confining all research to in vitro analysis of ABC transporters in detergent solutions or embedded in membrane-mimicking environments. Therefore, a largely unanswered question is how ABC exporters operate in situ, namely in the native membrane context of a metabolically active cell. This review focuses on novel mechanistic insights into type I ABC exporters gained through a unique combination of structure determination, biochemical characterization, generation of conformation-specific nanobodies/sybodies and double electron-electron resonance.

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Generation of synthetic nanobodies against delicate proteins

Cited by 134 publications

References 58 publications

Liquid drop of DNA libraries reveals total genome information

Liquid drop of DNA libraries reveals total genome information

A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

From in vitro towards in situ: structure‐based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

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