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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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Sequences of permuted TnAK from the unselected and selected libraries. (A) Color coding of the core (blue) AMP binding (red) and lid (green) domains in TnAK sequence helps the reader map the location of new N- and C-termini in the permuted variants. (B) The TnAK residues encoded by the first codon in unselected adk are indicated with a line, as well as (C) selected variants that complement E. coli CV2. (D) The TnAK residues encoded by the first codon in each functional variant (yellow spheres) are mapped onto the structure of Bacillus subtilis AK (36), which displays 48% sequence identity with TnAK. The substrate analog P1,P5-di(adenosine-5) pentaphosphate is shown in magenta. The image was created using PyMOL.
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gks060-F4: Sequences of permuted TnAK from the unselected and selected libraries. (A) Color coding of the core (blue) AMP binding (red) and lid (green) domains in TnAK sequence helps the reader map the location of new N- and C-termini in the permuted variants. (B) The TnAK residues encoded by the first codon in unselected adk are indicated with a line, as well as (C) selected variants that complement E. coli CV2. (D) The TnAK residues encoded by the first codon in each functional variant (yellow spheres) are mapped onto the structure of Bacillus subtilis AK (36), which displays 48% sequence identity with TnAK. The substrate analog P1,P5-di(adenosine-5) pentaphosphate is shown in magenta. The image was created using PyMOL.

Mentions: To evaluate how the diversity of vectors created by PERMUTE relates to the TnAK domain structure (Figure 4A), we sequenced individual clones from our final unselected library. Figure 4B shows that among the vectors successfully sequenced (nā€‰=ā€‰55), unique permuted adk were discovered whose first codon corresponds to residues within all three domains (AMP binding, core and lid) of the parental TnAK. Insertion sites were also observed in all three possible codon frames (with 21% in frame and 79% out of frame) in these unselected variants. In addition, minitransposons were observed in both possible orientations relative to the permuted adk genes (Figure 2B), with 36% inserted in the orientation that allows for productive transcription of permuted variants and 64% in the opposite orientation.Figure 4.


A transposase strategy for creating libraries of circularly permuted proteins.

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

Sequences of permuted TnAK from the unselected and selected libraries. (A) Color coding of the core (blue) AMP binding (red) and lid (green) domains in TnAK sequence helps the reader map the location of new N- and C-termini in the permuted variants. (B) The TnAK residues encoded by the first codon in unselected adk are indicated with a line, as well as (C) selected variants that complement E. coli CV2. (D) The TnAK residues encoded by the first codon in each functional variant (yellow spheres) are mapped onto the structure of Bacillus subtilis AK (36), which displays 48% sequence identity with TnAK. The substrate analog P1,P5-di(adenosine-5) pentaphosphate is shown in magenta. The image was created using PyMOL.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks060-F4: Sequences of permuted TnAK from the unselected and selected libraries. (A) Color coding of the core (blue) AMP binding (red) and lid (green) domains in TnAK sequence helps the reader map the location of new N- and C-termini in the permuted variants. (B) The TnAK residues encoded by the first codon in unselected adk are indicated with a line, as well as (C) selected variants that complement E. coli CV2. (D) The TnAK residues encoded by the first codon in each functional variant (yellow spheres) are mapped onto the structure of Bacillus subtilis AK (36), which displays 48% sequence identity with TnAK. The substrate analog P1,P5-di(adenosine-5) pentaphosphate is shown in magenta. The image was created using PyMOL.
Mentions: To evaluate how the diversity of vectors created by PERMUTE relates to the TnAK domain structure (Figure 4A), we sequenced individual clones from our final unselected library. Figure 4B shows that among the vectors successfully sequenced (nā€‰=ā€‰55), unique permuted adk were discovered whose first codon corresponds to residues within all three domains (AMP binding, core and lid) of the parental TnAK. Insertion sites were also observed in all three possible codon frames (with 21% in frame and 79% out of frame) in these unselected variants. In addition, minitransposons were observed in both possible orientations relative to the permuted adk genes (Figure 2B), with 36% inserted in the orientation that allows for productive transcription of permuted variants and 64% in the opposite orientation.Figure 4.

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.

Show MeSH
Related in: MedlinePlus