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A transposase strategy for creating libraries of circularly permuted proteins.

Mehta MM, Liu S, Silberg JJ - Nucleic Acids Res. (2012)

Bottom Line: In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted.Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods.In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology Rice University, Houston, TX 77251, USA.

ABSTRACT
A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.

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Vector types present in a PERMUTE library. (A) The minitransposon can be integrated in two orientations within the target vector, only one of which is expected to transcribe the permuted adk genes (top) using the promoter Pc. (B) Among the vectors that transcribe permuted adk, one-third are predicted to have their codons (NNN and nnn) in frame such that they translate a circularly permuted TnAK. The initial five base pairs of each permuted gene (yellow) are duplicated and fused to the 3′-end of each gene by the minitransposon integration reaction. The first four base pairs of the integrated minitransposon (Figure 1C) that become fused to circularly permuted genes are shown in bold, illustrating how the stop codon (TGA) is only in frame within the vectors that transcribe and translate circularly permuted proteins.
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gks060-F2: Vector types present in a PERMUTE library. (A) The minitransposon can be integrated in two orientations within the target vector, only one of which is expected to transcribe the permuted adk genes (top) using the promoter Pc. (B) Among the vectors that transcribe permuted adk, one-third are predicted to have their codons (NNN and nnn) in frame such that they translate a circularly permuted TnAK. The initial five base pairs of each permuted gene (yellow) are duplicated and fused to the 3′-end of each gene by the minitransposon integration reaction. The first four base pairs of the integrated minitransposon (Figure 1C) that become fused to circularly permuted genes are shown in bold, illustrating how the stop codon (TGA) is only in frame within the vectors that transcribe and translate circularly permuted proteins.

Mentions: For a target gene encoding a protein of length N, PERMUTE generates up to 6N unique vectors. This occurs because the synthetic minitransposon can be integrated into the target gene in two orientations after each base pair (Figure 2A). Only one of these orientations has the minitransposon oriented so that the target gene is transcribed. The minitransposon can also be integrated at different locations within each codon of the target gene (Figure 2B). Among the vectors with the minitransposon integrated in an orientation that leads to transcription of the target gene, only one-third of the possible vectors have a minitransposon integrated in the codon frame and orientation that leads to translation of a circularly permuted protein. This subset of vectors expresses permuted proteins with a peptide (MGFRIYRETLSRFSCAAQ) fused to their N-terminus, because translation is initiated within the minitransposon before the MuA-binding site (R2R1) that precedes the permuted gene. Two residues are also added to the C-terminus of these circularly permuted proteins, whose identity depends on the location of minitransposon integration within the original gene. Among the other vectors that transcribe the permuted gene, the target gene is out of frame with respect to the start codon. A majority of these vectors are not expected to express a circularly permuted protein. However, some of these vectors could express portions of the target protein using alternative start codons or translational frameshifting (34).Figure 2.


A transposase strategy for creating libraries of circularly permuted proteins.

Mehta MM, Liu S, Silberg JJ - Nucleic Acids Res. (2012)

Vector types present in a PERMUTE library. (A) The minitransposon can be integrated in two orientations within the target vector, only one of which is expected to transcribe the permuted adk genes (top) using the promoter Pc. (B) Among the vectors that transcribe permuted adk, one-third are predicted to have their codons (NNN and nnn) in frame such that they translate a circularly permuted TnAK. The initial five base pairs of each permuted gene (yellow) are duplicated and fused to the 3′-end of each gene by the minitransposon integration reaction. The first four base pairs of the integrated minitransposon (Figure 1C) that become fused to circularly permuted genes are shown in bold, illustrating how the stop codon (TGA) is only in frame within the vectors that transcribe and translate circularly permuted proteins.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3351165&req=5

gks060-F2: Vector types present in a PERMUTE library. (A) The minitransposon can be integrated in two orientations within the target vector, only one of which is expected to transcribe the permuted adk genes (top) using the promoter Pc. (B) Among the vectors that transcribe permuted adk, one-third are predicted to have their codons (NNN and nnn) in frame such that they translate a circularly permuted TnAK. The initial five base pairs of each permuted gene (yellow) are duplicated and fused to the 3′-end of each gene by the minitransposon integration reaction. The first four base pairs of the integrated minitransposon (Figure 1C) that become fused to circularly permuted genes are shown in bold, illustrating how the stop codon (TGA) is only in frame within the vectors that transcribe and translate circularly permuted proteins.
Mentions: For a target gene encoding a protein of length N, PERMUTE generates up to 6N unique vectors. This occurs because the synthetic minitransposon can be integrated into the target gene in two orientations after each base pair (Figure 2A). Only one of these orientations has the minitransposon oriented so that the target gene is transcribed. The minitransposon can also be integrated at different locations within each codon of the target gene (Figure 2B). Among the vectors with the minitransposon integrated in an orientation that leads to transcription of the target gene, only one-third of the possible vectors have a minitransposon integrated in the codon frame and orientation that leads to translation of a circularly permuted protein. This subset of vectors expresses permuted proteins with a peptide (MGFRIYRETLSRFSCAAQ) fused to their N-terminus, because translation is initiated within the minitransposon before the MuA-binding site (R2R1) that precedes the permuted gene. Two residues are also added to the C-terminus of these circularly permuted proteins, whose identity depends on the location of minitransposon integration within the original gene. Among the other vectors that transcribe the permuted gene, the target gene is out of frame with respect to the start codon. A majority of these vectors are not expected to express a circularly permuted protein. However, some of these vectors could express portions of the target protein using alternative start codons or translational frameshifting (34).Figure 2.

Bottom Line: In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted.Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods.In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology Rice University, Houston, TX 77251, USA.

ABSTRACT
A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.

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Related in: MedlinePlus