Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector


Autoria(s): Khan, Seema; Hsu, Rachel; Jones, Alun; Ross, Ian L.; Hart, Derek N.J.; Kato, Masato
Contribuinte(s)

R. R. Burgess

Data(s)

01/09/2006

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

http://espace.library.uq.edu.au/view/UQ:80435

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