Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins

Vida, István; Fazekas, Zsolt; Gyulai, Gergö; Nagy-Fazekas, Dóra; Pálfy, Gyula; Stráner, Pál; Kiss, Éva; Perczel, András

doi:10.1111/1751-7915.13778

Cited by 5 publications

(2 citation statements)

References 28 publications

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“…An additional goal of the project was to create a suitable expression system to produce a biologically active antigen with good yield for testing the neutralizing nanobodies in a fast and accurate way. Although the most convenient method of recombinant protein production is rapid and economical bacterial production [ 28 , 29 ], efficiently expressing the RBD is challenging. The RBD consists of β-sheets and five disulfide bonds stabilizing its structure [ 30 , 31 ].…”

Section: Resultsmentioning

confidence: 99%

A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System

et al. 2023

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Antibodies are key proteins of the immune system, and they are widely used for both research and theragnostic applications. Among them, camelid immunoglobulins (IgG) differ from the canonical human IgG molecules, as their light chains are completely missing; thus, they have only variable domains on their heavy chains (VHHs). A single VHH domain, often called a nanobody, has favorable structural, biophysical, and functional features compared to canonical antibodies. Therefore, robust and efficient production protocols relying on recombinant technologies are in high demand. Here, by utilizing ecotin, an Escherichia coli protein, as a fusion partner, we present a bacterial expression system that allows an easy, fast, and cost-effective way to prepare nanobodies. Ecotin was used here as a periplasmic translocator and a passive refolding chaperone, which allowed us to reach high-yield production of nanobodies. We also present a new, easily applicable prokaryotic expression and purification method of the receptor-binding domain (RBD) of the SARS-CoV-2 S protein for interaction assays. We demonstrate using ECD spectroscopy that the bacterially produced RBD is well-folded. The bacterially produced nanobody was shown to bind strongly to the recombinant RBD, with a Kd of 10 nM. The simple methods presented here could facilitate rapid interaction measurements in the event of the appearance of additional SARS-CoV-2 variants.

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Section: Resultsmentioning

confidence: 99%

A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System

et al. 2023

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“…(Budapest, Hungary). Exendin-4 (or Exenatide) derivative miniprotein (EEEAVRLYIQWLKEGGPSSGRPPPS) (a marketed drug for type-2 diabetes mellitus, E5) was produced by bacterial expression using the previously described protocol [ 57 ]. Specific parameters of the proteins were summarized in Table 5 and Table S1 .…”

Section: Methodsmentioning

confidence: 99%

Comparative Study of the Solid-Liquid Interfacial Adsorption of Proteins in Their Native and Amyloid Forms

Ábrahám

Massignan

Gyulai

et al. 2022

IJMS

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The adhesive properties of amyloid fibers are thought to play a crucial role in various negative and positive aggregation processes, the study of which might help in their understanding and control. Amyloids have been prepared from two proteins, lysozyme and β-lactoglobulin, as well as an Exendin-4 derivative miniprotein (E5). Thermal treatment was applied to form amyloids and their structure was verified by thioflavin T (ThT), 8-Anilino-1-naphthalenesulfonic acid (ANS) dye tests and electronic circular dichroism spectroscopy (ECD). Adsorption properties of the native and amyloid forms of the three proteins were investigated and compared using the mass-sensitive quartz crystal microbalance (QCM) technique. Due to the possible electrostatic and hydrophobic interactions, similar adsorbed amounts were found for the native or amyloid forms, while the structures of the adsorbed layers differed significantly. Native proteins formed smooth and dense adsorption layers. On the contrary, a viscoelastic, highly loose layer was formed in the presence of the amyloid forms, shown by increased motional resistance values determined by the QCM technique and also indicated by atomic force microscopy (AFM) and wettability measurements. The elongated structure and increased hydrophobicity of amyloids might contribute to this kind of aggregation.

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Target‐Templated Construction of Functional Proteomimetics Using Photo‐Foldamer Libraries

Wéber,

Ábrányi‐Balogh,

Nagymihály

et al. 2024

Angewandte Chemie

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Current methods for proteomimetic engineering rely on structure‐based design. Here we describe a design strategy that allows the construction of proteomimetics against challenging targets without a priori characterization of the target surface. Our approach relies on (i) a 100‐membered photoreactive foldamer library, the members of which act as local surface mimetics, and (ii) the subsequent affinity maturation of the primary hits using systems chemistry. Two surface‐oriented proteinogenic side chains drove the interactions between the short helical foldamer fragments and the proteins. Diazirine‐based photo‐crosslinking was applied to sensitively detected and localize binding even to shallow and dynamic patches on representatively difficult targets. Photo‐foldamers identified functionally relevant protein interfaces, allosteric and previously unexplored targetable regions on the surface of STAT3 and an oncogenic K‐Ras variant. Target‐templated dynamic linking of foldamer hits resulted in two orders of magnitude affinity improvement in a single step. The dimeric K‐Ras ligand mimicked protein‐like catalytic functions. The photo‐foldamer approach thus enables the highly efficient mapping of protein‐protein interaction sites and provides a viable starting point for proteomimetic ligand development without a priori structural hypotheses.

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Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins

Cited by 5 publications

References 28 publications

A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System

A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System

Comparative Study of the Solid-Liquid Interfacial Adsorption of Proteins in Their Native and Amyloid Forms

Target‐Templated Construction of Functional Proteomimetics Using Photo‐Foldamer Libraries

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