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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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Temperature selection of the target vector. (A) Cells transformed with the target vector (pMM1) complement bacterial growth at 30°C but not 43°C on LB agar medium containing chloramphenicol (34 µg/ml). (B) NotI treatment of the target vector before and after performing the MuA reaction. For MuA reaction products, NotI digestion was performed on DNA that was purified from E. coli that had been selected for growth at 30 or 43°C on LB agar plates containing kanamycin (25 µg/ml) and chloramphenicol (15 µg/ml). NotI cleaved the target vector into two products: adk (669 bp) and target vector backbone (2745 bp). In contrast, NotI digestion of MuA reaction products amplified in E. coli at 30 and 43°C yielded four products (asterisk), whose weights correspond to adk (669 bp), adk with a single integrated minitransposon (2483 bp), target vector backbone (2745 bp) and target vector backbone containing a single integrated minitransposon (4559). A band corresponding to the minitransposon alone (1809 bp) was not observed.
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gks060-F3: Temperature selection of the target vector. (A) Cells transformed with the target vector (pMM1) complement bacterial growth at 30°C but not 43°C on LB agar medium containing chloramphenicol (34 µg/ml). (B) NotI treatment of the target vector before and after performing the MuA reaction. For MuA reaction products, NotI digestion was performed on DNA that was purified from E. coli that had been selected for growth at 30 or 43°C on LB agar plates containing kanamycin (25 µg/ml) and chloramphenicol (15 µg/ml). NotI cleaved the target vector into two products: adk (669 bp) and target vector backbone (2745 bp). In contrast, NotI digestion of MuA reaction products amplified in E. coli at 30 and 43°C yielded four products (asterisk), whose weights correspond to adk (669 bp), adk with a single integrated minitransposon (2483 bp), target vector backbone (2745 bp) and target vector backbone containing a single integrated minitransposon (4559). A band corresponding to the minitransposon alone (1809 bp) was not observed.

Mentions: We tested PERMUTE by applying it to AK from Thermotoga neapolitana (33), a thermostable AK whose phosphotransferase activity (ADP + ADP ⇔ AMP + ATP) can be assessed using E. coli complementation (35). To obtain a target vector for performing PERMUTE, we cloned the gene encoding TnAK into a vector with a temperature-sensitive origin of replication (32). Figure 3A shows that the target vector containing the TnAK gene complements E. coli growth on LB agar medium containing chloramphenicol at 30°C but not at 43°C. To obtain an ensemble of target vectors containing an integrated minitransposon, this target vector was incubated with MuA and the synthetic minitransposon (Figure 1C), the DNA products of this reaction were transformed in E. coli and cells were grown on LB agar plates containing chloramphenicol and kanamycin at a temperature (43°C) where the target vector does not confer resistance to chloramphenicol. Plating two-thirds of a single transformation reaction yielded approximately 9000 colonies. Assuming the cells doubled once during the outgrowth after transformation, simulation of our procedure estimated that our single transformation sampled 6750 variants. This number is greater than the total number of possible vectors that can be created by random minitransposon insertion into the target vector (n = 5500), which we calculated as the number of possible insertion sites in the target vector that do not disrupt chloramphenicol resistance times the number of possible minitransposon insertion orientations at each site. Using these values for sample size and number of possible unique variants, we estimated that our reaction sampled 71% of the permuted AK variants. Furthermore, we calculated that this sampling could be increased to 91% by simply running two insertion reactions in parallel and >99% by running four reactions in parallel.Figure 3.


A transposase strategy for creating libraries of circularly permuted proteins.

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

Temperature selection of the target vector. (A) Cells transformed with the target vector (pMM1) complement bacterial growth at 30°C but not 43°C on LB agar medium containing chloramphenicol (34 µg/ml). (B) NotI treatment of the target vector before and after performing the MuA reaction. For MuA reaction products, NotI digestion was performed on DNA that was purified from E. coli that had been selected for growth at 30 or 43°C on LB agar plates containing kanamycin (25 µg/ml) and chloramphenicol (15 µg/ml). NotI cleaved the target vector into two products: adk (669 bp) and target vector backbone (2745 bp). In contrast, NotI digestion of MuA reaction products amplified in E. coli at 30 and 43°C yielded four products (asterisk), whose weights correspond to adk (669 bp), adk with a single integrated minitransposon (2483 bp), target vector backbone (2745 bp) and target vector backbone containing a single integrated minitransposon (4559). A band corresponding to the minitransposon alone (1809 bp) was not observed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351165&req=5

gks060-F3: Temperature selection of the target vector. (A) Cells transformed with the target vector (pMM1) complement bacterial growth at 30°C but not 43°C on LB agar medium containing chloramphenicol (34 µg/ml). (B) NotI treatment of the target vector before and after performing the MuA reaction. For MuA reaction products, NotI digestion was performed on DNA that was purified from E. coli that had been selected for growth at 30 or 43°C on LB agar plates containing kanamycin (25 µg/ml) and chloramphenicol (15 µg/ml). NotI cleaved the target vector into two products: adk (669 bp) and target vector backbone (2745 bp). In contrast, NotI digestion of MuA reaction products amplified in E. coli at 30 and 43°C yielded four products (asterisk), whose weights correspond to adk (669 bp), adk with a single integrated minitransposon (2483 bp), target vector backbone (2745 bp) and target vector backbone containing a single integrated minitransposon (4559). A band corresponding to the minitransposon alone (1809 bp) was not observed.
Mentions: We tested PERMUTE by applying it to AK from Thermotoga neapolitana (33), a thermostable AK whose phosphotransferase activity (ADP + ADP ⇔ AMP + ATP) can be assessed using E. coli complementation (35). To obtain a target vector for performing PERMUTE, we cloned the gene encoding TnAK into a vector with a temperature-sensitive origin of replication (32). Figure 3A shows that the target vector containing the TnAK gene complements E. coli growth on LB agar medium containing chloramphenicol at 30°C but not at 43°C. To obtain an ensemble of target vectors containing an integrated minitransposon, this target vector was incubated with MuA and the synthetic minitransposon (Figure 1C), the DNA products of this reaction were transformed in E. coli and cells were grown on LB agar plates containing chloramphenicol and kanamycin at a temperature (43°C) where the target vector does not confer resistance to chloramphenicol. Plating two-thirds of a single transformation reaction yielded approximately 9000 colonies. Assuming the cells doubled once during the outgrowth after transformation, simulation of our procedure estimated that our single transformation sampled 6750 variants. This number is greater than the total number of possible vectors that can be created by random minitransposon insertion into the target vector (n = 5500), which we calculated as the number of possible insertion sites in the target vector that do not disrupt chloramphenicol resistance times the number of possible minitransposon insertion orientations at each site. Using these values for sample size and number of possible unique variants, we estimated that our reaction sampled 71% of the permuted AK variants. Furthermore, we calculated that this sampling could be increased to 91% by simply running two insertion reactions in parallel and >99% by running four reactions in parallel.Figure 3.

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