Functional expression of secreted proteins from a bicistronic retroviral cassette based on foot-and-mouth disease virus 2A can be position dependent.
The expression of two or more genes from a single viral vector has been widely used to label or select for cells containing the transgenic element. Identification of the foot-and-mouth disease virus (FMDV) 2A cleavage peptide as a polycistronic linker capable of producing equivalent levels of transgene expression has greatly improved this approach in the field of gene therapy. However, as a consequence of 2A posttranslational cleavage the upstream protein is left with a residual 19 amino acids from the 2A sequence on its carboxy terminus, and the downstream protein is left with an additional 2 to 5 amino acids on its amino terminus. Here we have assessed the functional consequences of the FMDV 2A cleavage motif on two secreted proteins (interleukin [IL]-2 and transforming growth factor [TGF]-β) when expressed from a retroviral bicistronic vector. Whereas IL-2 expression and function were found to be unaffected by the 2A motif in either orientation, functional expression of secreted TGF-β was significantly abrogated when the transgene was expressed upstream of the 2A sequence. We believe this is a consequence of aberrant cleavage and intracellular trafficking of the TGF-β polyprotein. These results highlight that to achieve functional expression of secreted proteins consideration must be taken of the transgenic protein's posttranslational modification and trafficking when using 2A-based bicistronic cassettes.
Rothwell
Dominic G
DG
Cancer Research UK Department of Medical Oncology, School of Cancer and Imaging Sciences, University of Manchester, Manchester Academic Health Science Centre, Christie NHS Trust, Manchester M20 4BX, United Kingdom. drothwell@picr.man.ac.uk
Crossley
Rachel
R
Bridgeman
John S
JS
Sheard
Victoria
V
Zhang
Yining
Y
Sharp
Tyson V
TV
Hawkins
Robert E
RE
Gilham
David E
DE
McKay
Tristan R
TR
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005536:Foot-and-Mouth Disease / Q000235:genetics; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011499:Protein Processing, Post-Translational; D012190:Retroviridae / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D007376:Interleukin-2; D016212:Transforming Growth Factor beta; D014764:Viral Proteins