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

Andrew S Flies; Jocelyn M Darby; Peter R Murphy; Terry L Pinfold; Amanda L Patchett; Patrick R Lennard

doi:10.21769/BioProtoc.3696

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

Bio Protoc. 2020 Jul 5;10(13):e3696. doi: 10.21769/BioProtoc.3696.

Authors

Andrew S Flies¹, Jocelyn M Darby¹, Peter R Murphy^{1

2}, Terry L Pinfold³, Amanda L Patchett¹, Patrick R Lennard^{1

4}

Affiliations

¹ Menzies Institute for Medical Research, College of Health and Medicine, University of Tasmania, Hobart, TAS 7000, Australia.
² University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, Australia.
³ School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, TAS 7000, Australia.
⁴ The Roslin Institute and Royal School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK.

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.

Keywords: Comparative immunology; Fluorescent; Fusion protein; Non-model organism; Recombinant; Soluble; Trans-endocytosis.