Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector
Contribuinte(s) |
R. R. Burgess |
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Data(s) |
01/09/2006
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Resumo |
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 (30bp 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. (c) 2006 Elsevier Inc. All rights reserved. |
Identificador | |
Idioma(s) |
eng |
Publicador |
Elsevier Science BV |
Palavras-Chave | #Recombinant Protein #Influenza Matrix Protein M1 #Alternative Initiation Codon #Antigen Presenting Cells #Biochemical Research Methods #Biochemistry & Molecular Biology #Biotechnology & Applied Microbiology #Recombination #Protein #Virus #C1 #320305 Medical Biochemistry - Proteins and Peptides #780105 Biological sciences |
Tipo |
Journal Article |