Inhibition of 2A-mediated 'cleavage' of certain artificial polyproteins bearing N-terminal signal sequences.

Biotechnology Journal
Pablo de FelipeM D Ryan

Abstract

Where 2A oligopeptide sequences occur within ORFs, the formation of the glycyl-prolyl peptide bond at the C-terminus of (each) 2A does not occur. This property can be used to concatenate sequences encoding several proteins into a single ORF: each component of such an artificial polyprotein is generated as a discrete translation product. 2A and '2A-like' sequences have become widely utilised in biotechnology and biomedicine. Individual proteins may also be co- and post-translationally targeted to a variety of sub-cellular sites. In the case of polyproteins bearing N-terminal signal sequences we observed, however, that the protein downstream of 2A (no signal) was translocated into the endoplasmic reticulum (ER). We interpreted these data as a form of 'slipstream' translocation: downstream proteins, without signals, were translocated through a translocon pore already formed by the signal sequence at the N-terminus of the polyprotein. Here we show this effect is, in fact, due to inhibition of the 2A reaction (formation of fusion protein) by the C-terminal region (immediately upstream of 2A) of some proteins when translocated into the ER. Solutions to this problem include the use of longer 2As (with a favourable upstream context) or...Continue Reading

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Citations

Apr 10, 2012·Journal of Virological Methods·Nuttee SureeDong Sung An
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Nov 19, 2020·International Journal of Molecular Sciences·Anne-Catherine PratsEric Lacazette

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Methods Mentioned

BETA
PCR
transfection
glycosylation
protein folding

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