Engineering and characterization of GFP-targeting nanobody: Expression, purification, and post-translational modification analysis.
Protein Expr Purif
; 221: 106501, 2024 Sep.
Article
en En
| MEDLINE
| ID: mdl-38782081
ABSTRACT
Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.
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Banco de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes de Fusión
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Procesamiento Proteico-Postraduccional
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Proteínas Fluorescentes Verdes
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Escherichia coli
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Anticuerpos de Dominio Único
Límite:
Humans
Idioma:
En
Año:
2024
Tipo del documento:
Article