A Step‐by‐Step Guide for the Production ...
Document type :
Article dans une revue scientifique: Article original
DOI :
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Title :
A Step‐by‐Step Guide for the Production of Recombinant Fluorescent TAT‐HA‐Tagged Proteins and their Transduction into Mammalian Cells
Author(s) :
Abou Anny, Christer [Auteur]
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576
Nouaille, Sébastien [Auteur]
Toulouse Biotechnology Institute [TBI]
Fauré, Régis [Auteur]
Toulouse Biotechnology Institute [TBI]
Schulz, Celine [Auteur]
Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 [UGSF]
Spriet, Corentin [Auteur]
Plateformes Lilloises en Biologie et Santé (PLBS) - UAR 2014 - US 41
Huvent, Isabelle [Auteur]
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576
Biot, Christophe [Auteur]
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576
Lefebvre, Tony [Auteur]
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576
Nouaille, Sébastien [Auteur]
Toulouse Biotechnology Institute [TBI]
Fauré, Régis [Auteur]
Toulouse Biotechnology Institute [TBI]
Schulz, Celine [Auteur]
Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 [UGSF]
Spriet, Corentin [Auteur]
Plateformes Lilloises en Biologie et Santé (PLBS) - UAR 2014 - US 41
Huvent, Isabelle [Auteur]
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576
Biot, Christophe [Auteur]
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576
Lefebvre, Tony [Auteur]
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576
Journal title :
Current Protocols
Abbreviated title :
Current Protocols
Volume number :
4
Publisher :
Wiley
Publication date :
2024-03
HAL domain(s) :
Sciences du Vivant [q-bio]
English abstract : [en]
AbstractInvestigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag ...
Show more >AbstractInvestigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS‐PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step‐by‐step the engineering of a recombinant protein with various tags: TAT‐HA (trans‐activator of transduction‐hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni‐nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT‐HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step‐by‐step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein productionBasic Protocol 2: Protein purificationBasic Protocol 3: Protein transduction in mammalian cellsShow less >
Show more >AbstractInvestigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS‐PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step‐by‐step the engineering of a recombinant protein with various tags: TAT‐HA (trans‐activator of transduction‐hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni‐nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT‐HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step‐by‐step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein productionBasic Protocol 2: Protein purificationBasic Protocol 3: Protein transduction in mammalian cellsShow less >
Language :
Anglais
Audience :
Internationale
Popular science :
Non
Administrative institution(s) :
Université de Lille
CNRS
CNRS
Collections :
Research team(s) :
O-GlcNAcylation, signalisation cellulaire et cycle cellulaire
Chemical Glycobiology
Chemical Glycobiology
Submission date :
2024-05-07T12:55:12Z
2024-05-15T08:10:34Z
2024-05-15T08:10:34Z
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