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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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Quantification of His-tagged AtAOS, PsLOX3 and MtHPLF proteins from bacterial lysates. Total proteins were extracted from cells producing WT and FS enzymes and were separated on a 4–12% polyacrylamide gradient SDS-PAGE gel. About 20 μg total proteins were stained with Coomassie blue (A) to check protein quantifications and loading levels. Lane M, molecular weight marker (SeeBlue Plus2; Invitrogen); lane 1 AtAOS wild type, lane 2 AtAOS frameshift; lane 3 MtHPLF wild type; lane 4 MtHPLF frameshift; lane 5 PsLOX3 wild type, lane 6 PsLOX3 frameshift. For western blot protein quantifications, 20 μg of total protein from cells expressing AtAOS or MtHPLF and 60 μg of total protein from cells expressing PsLOX3 were transferred to a nitrocellulose membrane and detected with a monoclonal antibody against the His tag (B) (see Materials and Methods for further details). (C) Digitally quantified band intensities from western blotting showing means and standard errors from three independent samples. Black bars—WT, grey bars—FS.
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Figure 3: Quantification of His-tagged AtAOS, PsLOX3 and MtHPLF proteins from bacterial lysates. Total proteins were extracted from cells producing WT and FS enzymes and were separated on a 4–12% polyacrylamide gradient SDS-PAGE gel. About 20 μg total proteins were stained with Coomassie blue (A) to check protein quantifications and loading levels. Lane M, molecular weight marker (SeeBlue Plus2; Invitrogen); lane 1 AtAOS wild type, lane 2 AtAOS frameshift; lane 3 MtHPLF wild type; lane 4 MtHPLF frameshift; lane 5 PsLOX3 wild type, lane 6 PsLOX3 frameshift. For western blot protein quantifications, 20 μg of total protein from cells expressing AtAOS or MtHPLF and 60 μg of total protein from cells expressing PsLOX3 were transferred to a nitrocellulose membrane and detected with a monoclonal antibody against the His tag (B) (see Materials and Methods for further details). (C) Digitally quantified band intensities from western blotting showing means and standard errors from three independent samples. Black bars—WT, grey bars—FS.

Mentions: To confirm functionality of the expressed proteins, we performed western blot semi-quantification with E. coli crude extracts using an anti-His tag antibody (Figure 3). This experiment gave similar results to the activity measurements described above: there were almost threefold (292%) higher quantities of AtAOS-WT protein expressed in E. coli cultures compared to the FS clone and the amount of MtHPLF-FS protein expressed was 49% higher than that of the WT clone, showing that the increased enzyme activity data correlated with increased protein amounts. However, the amount of PsLOX3 was 74% higher in the WT compared to the FS cultures even though the activity measurements for these clones were almost the same. This latter result may be due to the lack of sensitivity of the spectrophotometric assay in measuring the relatively low LOX activity. The important point is that all three frameshift cDNAs produced correct-sized, active enzymes. Based on the western data the frameshifting efficiencies are approximately 60% (MtHPLF), 36% (PsLOX) and 25% (AtAOS).Figure 3.


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)

Quantification of His-tagged AtAOS, PsLOX3 and MtHPLF proteins from bacterial lysates. Total proteins were extracted from cells producing WT and FS enzymes and were separated on a 4–12% polyacrylamide gradient SDS-PAGE gel. About 20 μg total proteins were stained with Coomassie blue (A) to check protein quantifications and loading levels. Lane M, molecular weight marker (SeeBlue Plus2; Invitrogen); lane 1 AtAOS wild type, lane 2 AtAOS frameshift; lane 3 MtHPLF wild type; lane 4 MtHPLF frameshift; lane 5 PsLOX3 wild type, lane 6 PsLOX3 frameshift. For western blot protein quantifications, 20 μg of total protein from cells expressing AtAOS or MtHPLF and 60 μg of total protein from cells expressing PsLOX3 were transferred to a nitrocellulose membrane and detected with a monoclonal antibody against the His tag (B) (see Materials and Methods for further details). (C) Digitally quantified band intensities from western blotting showing means and standard errors from three independent samples. Black bars—WT, grey bars—FS.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 3: Quantification of His-tagged AtAOS, PsLOX3 and MtHPLF proteins from bacterial lysates. Total proteins were extracted from cells producing WT and FS enzymes and were separated on a 4–12% polyacrylamide gradient SDS-PAGE gel. About 20 μg total proteins were stained with Coomassie blue (A) to check protein quantifications and loading levels. Lane M, molecular weight marker (SeeBlue Plus2; Invitrogen); lane 1 AtAOS wild type, lane 2 AtAOS frameshift; lane 3 MtHPLF wild type; lane 4 MtHPLF frameshift; lane 5 PsLOX3 wild type, lane 6 PsLOX3 frameshift. For western blot protein quantifications, 20 μg of total protein from cells expressing AtAOS or MtHPLF and 60 μg of total protein from cells expressing PsLOX3 were transferred to a nitrocellulose membrane and detected with a monoclonal antibody against the His tag (B) (see Materials and Methods for further details). (C) Digitally quantified band intensities from western blotting showing means and standard errors from three independent samples. Black bars—WT, grey bars—FS.
Mentions: To confirm functionality of the expressed proteins, we performed western blot semi-quantification with E. coli crude extracts using an anti-His tag antibody (Figure 3). This experiment gave similar results to the activity measurements described above: there were almost threefold (292%) higher quantities of AtAOS-WT protein expressed in E. coli cultures compared to the FS clone and the amount of MtHPLF-FS protein expressed was 49% higher than that of the WT clone, showing that the increased enzyme activity data correlated with increased protein amounts. However, the amount of PsLOX3 was 74% higher in the WT compared to the FS cultures even though the activity measurements for these clones were almost the same. This latter result may be due to the lack of sensitivity of the spectrophotometric assay in measuring the relatively low LOX activity. The important point is that all three frameshift cDNAs produced correct-sized, active enzymes. Based on the western data the frameshifting efficiencies are approximately 60% (MtHPLF), 36% (PsLOX) and 25% (AtAOS).Figure 3.

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