Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein.
We describe the construction of a plasmid (pCAT2AGUS) encoding a polyprotein in which a 19 amino acid sequence spanning the 2A region of the foot-and-mouth disease virus (FMDV) polyprotein was inserted between the reporter genes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS) maintaining a single, long open reading frame. Analysis of translation reactions programmed by this construct showed that the inserted FMDV sequence functioned in a manner similar to that observed in FMDV polyprotein processing: the CAT2AGUS polyprotein underwent a cotranslational, apparently autoproteolytic, cleavage yielding CAT-2A and GUS. Analysis of translation products derived from a series of constructs in which sequences were progressively deleted from the N-terminal region of the FMDV 2A insertion showed that cleavage required a minimum of 13 residues. The FMDV 2A sequence therefore provides the opportunity to engineer either whole proteins or domains such that they are cleaved apart cotranslationally with high efficiency.
Ryan
M D
MD
AFRC Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, UK.
Drew
J
J
D000595:Amino Acid Sequence; D000818:Animals; D005537:Aphthovirus / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004279:DNA, Viral; D005966:Glucuronidase / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D010455:Peptides; D014764:Viral Proteins; C057421:virus protein 2A; D015500:Chloramphenicol O-Acetyltransferase; D005966:Glucuronidase