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The gateway pDEST17 expression vector encodes a -1 ribosomal frameshifting sequence.

Belfield EJ, Hughes RK, Tsesmetzis N, Naldrett MJ, Casey R - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. eric.belfield@bbsrc.ac.uk

ABSTRACT
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.

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Related in: MedlinePlus

Time course of AtAOS, PsLOX3 and MtHPLF WT- and FS- specific activities as a function of time after induction. Escherichia. coli BL21 (DE3) transformed with pDEST17 AtAOS-WT (blue line), AtAOS-WT-FS (pink line), MtHPLF-WT (purple line), MtHPLF-FS (brown line), PsLOX3-WT (yellow line) and PsLOX3-FS (cyan line) clones were induced with 1 mM IPTG and cell samples collected at the indicated times. Enzyme activities were assayed as described in the Materials and methods section.
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Figure 2: Time course of AtAOS, PsLOX3 and MtHPLF WT- and FS- specific activities as a function of time after induction. Escherichia. coli BL21 (DE3) transformed with pDEST17 AtAOS-WT (blue line), AtAOS-WT-FS (pink line), MtHPLF-WT (purple line), MtHPLF-FS (brown line), PsLOX3-WT (yellow line) and PsLOX3-FS (cyan line) clones were induced with 1 mM IPTG and cell samples collected at the indicated times. Enzyme activities were assayed as described in the Materials and methods section.

Mentions: Enzymatic assays of the crude MtHPLF-FS E. coli extracts showed that a fully functional protein was being produced, even though a truncated peptide of 35 amino acids (aa) (3.9 kDa), 469 aa shorter than the wild-type MtHPLF-WT protein, was predicted. Kinetic data using the substrate 13-HPOT (Figure 2) showed the specific activity after 24-h induction was over twice as high (11.84 µmol/min/mg protein versus 5.05 µmol/min/mg protein) for the MtHPLF-FS frameshift mutant expression clone compared to the wild-type MtHPLF-WT.Figure 2.


The gateway pDEST17 expression vector encodes a -1 ribosomal frameshifting sequence.

Belfield EJ, Hughes RK, Tsesmetzis N, Naldrett MJ, Casey R - Nucleic Acids Res. (2007)

Time course of AtAOS, PsLOX3 and MtHPLF WT- and FS- specific activities as a function of time after induction. Escherichia. coli BL21 (DE3) transformed with pDEST17 AtAOS-WT (blue line), AtAOS-WT-FS (pink line), MtHPLF-WT (purple line), MtHPLF-FS (brown line), PsLOX3-WT (yellow line) and PsLOX3-FS (cyan line) clones were induced with 1 mM IPTG and cell samples collected at the indicated times. Enzyme activities were assayed as described in the Materials and methods section.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851650&req=5

Figure 2: Time course of AtAOS, PsLOX3 and MtHPLF WT- and FS- specific activities as a function of time after induction. Escherichia. coli BL21 (DE3) transformed with pDEST17 AtAOS-WT (blue line), AtAOS-WT-FS (pink line), MtHPLF-WT (purple line), MtHPLF-FS (brown line), PsLOX3-WT (yellow line) and PsLOX3-FS (cyan line) clones were induced with 1 mM IPTG and cell samples collected at the indicated times. Enzyme activities were assayed as described in the Materials and methods section.
Mentions: Enzymatic assays of the crude MtHPLF-FS E. coli extracts showed that a fully functional protein was being produced, even though a truncated peptide of 35 amino acids (aa) (3.9 kDa), 469 aa shorter than the wild-type MtHPLF-WT protein, was predicted. Kinetic data using the substrate 13-HPOT (Figure 2) showed the specific activity after 24-h induction was over twice as high (11.84 µmol/min/mg protein versus 5.05 µmol/min/mg protein) for the MtHPLF-FS frameshift mutant expression clone compared to the wild-type MtHPLF-WT.Figure 2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. eric.belfield@bbsrc.ac.uk

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus