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Functionalized Polysaccharides Improve Sensitivity of Tyramide/Peroxidase Proximity Labeling Assays through Electrostatic Interactions.
Heiniger, Malvina; Vanella, Rosario; Walsh-Korb, Zarah; Nash, Michael A.
Afiliación
  • Heiniger M; Department of Chemistry, University of Basel, Mattenstrasse 22, Basel 4058, Switzerland.
  • Vanella R; Department of Chemistry, University of Basel, Mattenstrasse 22, Basel 4058, Switzerland.
  • Walsh-Korb Z; Department of Chemistry, University of Basel, Mattenstrasse 22, Basel 4058, Switzerland.
  • Nash MA; Department of Chemistry, University of Basel, Mattenstrasse 22, Basel 4058, Switzerland.
ACS Biomater Sci Eng ; 10(9): 5869-5880, 2024 Sep 09.
Article en En | MEDLINE | ID: mdl-39121180
ABSTRACT
High-throughput assays that efficiently link genotype and phenotype with high fidelity are key to successful enzyme engineering campaigns. Among these assays, the tyramide/peroxidase proximity labeling method converts the product of an enzymatic reaction of a surface expressed enzyme to a highly reactive fluorescent radical, which labels the cell surface. In this context, maintaining the proximity of the readout reagents to the cell surface is crucial to prevent crosstalk and ensure that short-lived radical species react before diffusing away. Here, we investigated improvements in tyramide/peroxidase proximity labeling for enzyme screening. We modified chitosan (Cs) chains with horseradish peroxidase (HRP) and evaluated the effects of these conjugates on the efficiency of proximity labeling reactions on yeast cells displaying d-amino acid oxidase. By tethering HRP to chitosan through different chemical approaches, we localized the auxiliary enzyme close to the cell surface and enhanced the sensitivity of tyramide-peroxidase labeling reactions. We found that immobilizing HRP onto chitosan through a 5 kDa PEG linker improved labeling sensitivity by over 3.5-fold for substrates processed with a low turnover rate (e.g., d-lysine), while the sensitivity of the labeling for high activity substrates (e.g., d-alanine) was enhanced by over 0.6-fold. Such improvements in labeling efficiency broaden the range of enzymes and conditions that can be studied and screened by tyramide/peroxidase proximity labeling.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Quitosano / Electricidad Estática / Peroxidasa de Rábano Silvestre Idioma: En Revista: ACS Biomater Sci Eng Año: 2024 Tipo del documento: Article País de afiliación: Suiza Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Quitosano / Electricidad Estática / Peroxidasa de Rábano Silvestre Idioma: En Revista: ACS Biomater Sci Eng Año: 2024 Tipo del documento: Article País de afiliación: Suiza Pais de publicación: Estados Unidos