Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector.
Gateway technology is a powerful system for converting a single entry vector into a wide variety of expression vectors. We expressed recombinant influenza matrix protein M1 (FMP), a potent antigen for cytotoxic T cells, using the Gateway vector pET-DEST42 containing the FMP cDNA, and purified the expressed FMP as a single 32 kDa recombinant protein. N-terminal and internal protein sequencing, however, showed that the recombinant FMP contained an extra 10 amino acids fused to the N-terminal of native FMP. Further investigation of the DNA sequence adjacent to the 5'-FMP cDNA indicated that the "TTG" in the attB1 site (30 bp upstream of the "ATG" in the 5'-FMP cDNA) behaved as a dominant translation start site, resulting in a 10 amino acid extension of the recombinant FMP. Thus, it is possible that recombinant proteins produced by this Gateway vector contain unexpected vector-derived peptides, which may affect experimental outcomes.
Khan
Seema
S
Dendritic Cell Program, Mater Medical Research Institute, Brisbane, Qld, Australia.
Hsu
Rachel
R
Jones
Alun
A
Ross
Ian L
IL
Hart
Derek N J
DN
Kato
Masato
M
D001483:Base Sequence; D018387:Codon, Initiator / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D018387:Codon, Initiator; D011994:Recombinant Proteins