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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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The pDEST17 frameshift region. (A) The predicted RNA and deduced amino acid sequences of the pDEST17 MtHPLF-WT clone. Single-letter abbreviations for amino acid residues are used. (B) The predicted RNA sequences of pDEST17 MtHPLF-FS and deduced amino acid sequences for both the 0 frame and the −1 frameshift transcripts. The MtHPLF-FS construct has a guanine nucleotide deletion three bases into the original MtHPLF cDNA sequence (underlined in (4A)), to repair ribosomal −1 frameshifting at the homopolymeric adenine sequence, AAA-AAA, and restores the MtHPLF 0 ORF (one amino acid into the HPL protein), yielding a product of approximately the same size as the WT fusion protein. (C) Diagrammatic representation of the predicted translated peptides from the pDEST17-expressed MtHPLF-FS RNAs and location of the frameshift region. The hatched box at the C-terminus of the ∼4-kDa product represents the amino acids MLPHQKPPQPTSP and the dotted box near the N-terminus of the ∼57-kDa product represents the amino acids RLNA. Solid grey MtHPLF protein sequence box not to scale.
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Figure 4: The pDEST17 frameshift region. (A) The predicted RNA and deduced amino acid sequences of the pDEST17 MtHPLF-WT clone. Single-letter abbreviations for amino acid residues are used. (B) The predicted RNA sequences of pDEST17 MtHPLF-FS and deduced amino acid sequences for both the 0 frame and the −1 frameshift transcripts. The MtHPLF-FS construct has a guanine nucleotide deletion three bases into the original MtHPLF cDNA sequence (underlined in (4A)), to repair ribosomal −1 frameshifting at the homopolymeric adenine sequence, AAA-AAA, and restores the MtHPLF 0 ORF (one amino acid into the HPL protein), yielding a product of approximately the same size as the WT fusion protein. (C) Diagrammatic representation of the predicted translated peptides from the pDEST17-expressed MtHPLF-FS RNAs and location of the frameshift region. The hatched box at the C-terminus of the ∼4-kDa product represents the amino acids MLPHQKPPQPTSP and the dotted box near the N-terminus of the ∼57-kDa product represents the amino acids RLNA. Solid grey MtHPLF protein sequence box not to scale.

Mentions: To verify directly that the ribosomal frameshifting had occurred at the AAA-AAA sequence in the RNA, the MtHPLF-FS and -WT proteins were over-expressed in E. coli and homogeneous proteins were obtained by FPLC (22). MALDI-ToF MS analysis of tryptic peptides from purified MtHPLF-FS and MtHPLF-WT revealed that most of the peptides aligned over the entire length of proteins, but information was apparently lacking from the N-termini. To determine precisely where the frameshift occurs, we obtained the unequivocal sequence of the first 21 N-terminal amino acids of MtHPLF-FS using Edman degradation. Residues 22–24 were not clear. In-source decay sequencing yielded residues 11–35, aiding the interpretation of that part of the sequence obtained by Edman sequencing that was less clear. Residues 1–19 corresponded to amino acids encoded by the pDEST17 vector. At residue 20, a strong signal for Ser was detected; this corresponds to the residue encoded by the −1 reading frame, followed by Arg and Leu that would be conventionally encoded after the leftward frameshift (Figure 4). Armed with the revised sequence, the masses of the N-terminal peptides were calculated and found to be present in the initial MALDI peptide mass fingerprint data.Figure 4.


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)

The pDEST17 frameshift region. (A) The predicted RNA and deduced amino acid sequences of the pDEST17 MtHPLF-WT clone. Single-letter abbreviations for amino acid residues are used. (B) The predicted RNA sequences of pDEST17 MtHPLF-FS and deduced amino acid sequences for both the 0 frame and the −1 frameshift transcripts. The MtHPLF-FS construct has a guanine nucleotide deletion three bases into the original MtHPLF cDNA sequence (underlined in (4A)), to repair ribosomal −1 frameshifting at the homopolymeric adenine sequence, AAA-AAA, and restores the MtHPLF 0 ORF (one amino acid into the HPL protein), yielding a product of approximately the same size as the WT fusion protein. (C) Diagrammatic representation of the predicted translated peptides from the pDEST17-expressed MtHPLF-FS RNAs and location of the frameshift region. The hatched box at the C-terminus of the ∼4-kDa product represents the amino acids MLPHQKPPQPTSP and the dotted box near the N-terminus of the ∼57-kDa product represents the amino acids RLNA. Solid grey MtHPLF protein sequence box not to scale.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 4: The pDEST17 frameshift region. (A) The predicted RNA and deduced amino acid sequences of the pDEST17 MtHPLF-WT clone. Single-letter abbreviations for amino acid residues are used. (B) The predicted RNA sequences of pDEST17 MtHPLF-FS and deduced amino acid sequences for both the 0 frame and the −1 frameshift transcripts. The MtHPLF-FS construct has a guanine nucleotide deletion three bases into the original MtHPLF cDNA sequence (underlined in (4A)), to repair ribosomal −1 frameshifting at the homopolymeric adenine sequence, AAA-AAA, and restores the MtHPLF 0 ORF (one amino acid into the HPL protein), yielding a product of approximately the same size as the WT fusion protein. (C) Diagrammatic representation of the predicted translated peptides from the pDEST17-expressed MtHPLF-FS RNAs and location of the frameshift region. The hatched box at the C-terminus of the ∼4-kDa product represents the amino acids MLPHQKPPQPTSP and the dotted box near the N-terminus of the ∼57-kDa product represents the amino acids RLNA. Solid grey MtHPLF protein sequence box not to scale.
Mentions: To verify directly that the ribosomal frameshifting had occurred at the AAA-AAA sequence in the RNA, the MtHPLF-FS and -WT proteins were over-expressed in E. coli and homogeneous proteins were obtained by FPLC (22). MALDI-ToF MS analysis of tryptic peptides from purified MtHPLF-FS and MtHPLF-WT revealed that most of the peptides aligned over the entire length of proteins, but information was apparently lacking from the N-termini. To determine precisely where the frameshift occurs, we obtained the unequivocal sequence of the first 21 N-terminal amino acids of MtHPLF-FS using Edman degradation. Residues 22–24 were not clear. In-source decay sequencing yielded residues 11–35, aiding the interpretation of that part of the sequence obtained by Edman sequencing that was less clear. Residues 1–19 corresponded to amino acids encoded by the pDEST17 vector. At residue 20, a strong signal for Ser was detected; this corresponds to the residue encoded by the −1 reading frame, followed by Arg and Leu that would be conventionally encoded after the leftward frameshift (Figure 4). Armed with the revised sequence, the masses of the N-terminal peptides were calculated and found to be present in the initial MALDI peptide mass fingerprint data.Figure 4.

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