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Protein coexpression using FMDV 2A : effect of “linker” residues

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BioMedResearchInter2013.pdf (2.588Mb)
Date
04/2013
Author
Minskaia, Ekaterina
Ryan, Martin D.
Keywords
Multiple proteins
Single vector
Foot-and-mouth disease virus
Open reading frames (ORFs)
Cistron
F2A
T2A
C-terminal sequence
Cloning strategies
Biomedical applications
Biotechnological applications
Mouth-disease virus
Ribosome entry sites
Embryonic stem-cells
Open reading frame
Retroviral vector
Signal sequences
Gene-expression
T-cells
Cleavage
Peptide
Q Science
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Abstract
Many biomedical applications absolutely require, or are substantially enhanced by, coexpression of multiple proteins from a single vector. Foot-and-mouth disease virus 2A (F2A) and “2A-like” sequences (e.g., Thosea asigna virus 2A; T2A) are used widely for this purpose since multiple proteins can be coexpressed by linking open reading frames (ORFs) to form a single cistron. The activity of F2A “cleavage” may, however, be compromised by both the use of shorter versions of F2A and the sequences (derived from multiple-purpose cloning sites) used to link F2A to the upstream protein. To characterise these effects, different lengths of F2A and T2A were inserted between green and cherry fluorescent proteins. Mutations were introduced in the linker region immediately upstream of both F2A- and T2A-based constructs and activities determined using both cell-free translation systems and transfected cells. In shorter versions of F2A, activity may be affected by both the C-terminal sequence of the protein upstream and, equally strikingly, the residues immediately upstream introduced during cloning. Mutations significantly improved activity for shorter versions of F2A but could decrease activity in the case of T2A. These data will aid the design of cloning strategies for the co-expression of multiple proteins in biomedical/biotechnological applications.
Citation
Minskaia , E & Ryan , M D 2013 , ' Protein coexpression using FMDV 2A : effect of “linker” residues ' , BioMed Research International . https://doi.org/10.1155/2013/291730
Publication
BioMed Research International
Status
Peer reviewed
DOI
https://doi.org/10.1155/2013/291730
ISSN
2314-6133
Type
Journal article
Rights
Copyright © 2013 Ekaterina Minskaia and Martin D. Ryan. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Description
This article was made open access through BIS OA funding. The research was supported by the MRC.
Collections
  • University of St Andrews Research
URI
http://hdl.handle.net/10023/3864

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