The gateway pDEST17 expression vector encodes a -1 ribosomal frameshifting sequence.
The attB1 site in the Gateway (Invitrogen) bacterial expression vector pDEST17, necessary for in vitro site-specific recombination, contains the sequence AAA-AAA. The sequence A-AAA-AAG within the Escherichia coli dnaX gene is recognized as 'slippery' and promotes -1 translational frameshifting. We show here, by expressing in E. coli several plant cDNAs with and without single nucleotide deletions close to the translation initiation codons, that pDEST17 is intrinsically susceptible to -1 ribosomal frameshifting at the sequence C-AAA-AAA. The deletion mutants produce correct-sized, active enzymes with a good correlation between enzyme amount and activity. We demonstrate unambiguously the frameshift through a combination of Edman degradation, MALDI-ToF mass fingerprint analysis of tryptic peptides and MALDI-ToF reflectron in-source decay (rISD) sequencing. The degree of frameshifting depends on the nature of the sequence being expressed and ranged from 25 to 60%. These findings suggest that caution should be exercised when employing pDEST17 for high-level protein expression and that the attB1 site has some potential as a tool for studying -1 frameshifting.
Belfield
Eric J
EJ
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. eric.belfield@bbsrc.ac.uk
Hughes
Richard K
RK
Tsesmetzis
Nicolas
N
Naldrett
Mike J
MJ
Casey
Rod
R
D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism; D001287:Attachment Sites, Microbiological; D001483:Base Sequence; D015153:Blotting, Western; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D003627:Data Interpretation, Statistical; D004926:Escherichia coli / Q000235:genetics; D018965:Frameshifting, Ribosomal; D015870:Gene Expression; D005822:Genetic Vectors; D016680:Genome, Bacterial; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism; D008084:Lipoxygenase / Q000235:genetics / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D018749:RNA, Plant / Q000737:chemistry; D017422:Sequence Analysis, DNA
D018749:RNA, Plant; D003577:Cytochrome P-450 Enzyme System; D008084:Lipoxygenase; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase