Random tag insertions by Transposon Integration mediated Mutagenesis (TIM).
Transposon Integration mediated Mutagenesis (TIM) is a broadly applicable tool for protein engineering. This method combines random integration of modified bacteriophage Mu transposons with their subsequent defined excision employing type IIS restriction endonuclease AarI. TIM enables deletion or insertion of an arbitrary number of bases at random positions, insertion of functional sequence tags at random positions, replacing randomly selected triplets by a specific codon (e.g. scanning) and site-saturation mutagenesis. As a proof of concept a transposon named GeneOpenerAarIKan was designed and employed to introduce 6xHis tags randomly into the esterase EstC from Burkholderia gladioli. A TIM library was screened with colony based assays for clones with an integrated 6xHis tag and for clones exhibiting esterase activity. The employed strategy enables the isolation of randomly tagged active enzymes in single mutagenesis experiments.
Hoeller
Brigitte M
BM
Research Centre Applied Biocatalysis, Petersgasse 14, 8010 Graz, Austria.
Reiter
Birgit
B
Abad
Sandra
S
Graze
Ina
I
Glieder
Anton
A
D001426:Bacterial Proteins / Q000235:genetics; D010583:Bacteriophage mu / Q000235:genetics; D001483:Base Sequence; D042725:Burkholderia gladioli / Q000201:enzymology / Q000235:genetics*; D004251:DNA Transposable Elements; D004950:Esterases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D015183:Restriction Mapping
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004950:Esterases