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Generation and testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins

Citation

Flies, AS and Darby, JM and Murphy, PR and Pinfold, TL and Patchett, AL and Lennard, PR, Generation and testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins, Bio-protocol, 10, (13) Article e3696. ISSN 2331-8325 (2020) [Refereed Article]

Copyright Statement

Copyright 2020 The Authors; exclusive licensee Bio-protocol LLC.

DOI: doi:10.21769/BioProtoc.3696

Abstract

This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live co-culture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five parts: (A) In silico characterization of the gene-of-interest (GOI) and protein-of-interest (POI); (B) design of the expression vector; (C) assembly of the expression vector; (D) transfection of a eukaryotic cell line with the expression vector; (E) testing of the recombinant protein. This extensive protocol can be completed with only polymerase chain reaction (PCR) and cell culture training. Additionally, each part of the protocol can be used independently.

Item Details

Item Type:Refereed Article
Keywords:fluorescent, fusion protein, recombinant, comparative immunology, non-model organism, soluble, trans-endocytosis, FAST protein, expression vector, methods, protocol
Research Division:Biological Sciences
Research Group:Zoology
Research Field:Animal immunology
Objective Division:Health
Objective Group:Clinical health
Objective Field:Clinical health not elsewhere classified
UTAS Author:Flies, AS (Dr Andy Flies)
UTAS Author:Darby, JM (Ms Jocelyn Darby)
UTAS Author:Murphy, PR (Mr Peter Murphy)
UTAS Author:Pinfold, TL (Dr Terry Pinfold)
UTAS Author:Patchett, AL (Dr Amanda Patchett)
UTAS Author:Lennard, PR (Mr Patrick Lennard)
ID Code:141667
Year Published:2020
Funding Support:Australian Research Council (DE180100484)
Web of Science® Times Cited:2
Deposited By:Menzies Institute for Medical Research
Deposited On:2020-11-09
Last Modified:2021-04-07
Downloads:0

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