Prokaryotic expression and purification of soluble maize Ac transposase.
Transposons are mobile genetic elements that are found in all eukaryotic and prokaryotic species studied to date. The Maize Activator (Ac) transposase recognizes and excises Ac and Dissociation (Ds) elements and mediates insertion elsewhere in the genome. Insertions of Ds can cause disruption in gene sequences and hence are important functional genomics tool for tagging and cloning of unknown gene sequences. The involvement of Ac transposase (AcTPase) in Ds movement is well documented; however, protein structure and function of AcTPase is poorly understood. To express the maize AcTPase in E. coli, Ac cDNA was synthesized with an N-terminal 6xHis tag and cloned in pTrcAc expression vector. The expression cassette was induced in Rosetta2 (DE3) E. coli lines. End-point RT-PCR confirmed the integrity of AcTPase mRNA during cell culture. Autoinducing cultures grown at 37 °C produced prominent partial AcTPase products of ~40 kDa and ~70 kDa. Trypsin digestion and mass spectrometry analyses confirmed AcTPase in both the eluted peptides. When the cultures were grown at 22-25 °C for 24 h the expected ~90 kDa AcTPase soluble product was detected. The successful expression of full length AcTPase in soluble form allows further investigation of its structure and function.
Plant Science Department, McGill University, Quebec, Canada. neil.lamb-palmer@mail.mcgill.ca
Neil Dylan
ND
Lamb-Palmer
Manjit
M
Singh
John P
JP
Dalton
Jaswinder
J
Singh
D002478:Cells, Cultured; D004251:DNA Transposable Elements / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D018745:Genome, Plant; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011387:Prokaryotic Cells / Q000378:metabolism*; D019895:Transposases / Q000235:genetics* / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D018076:DNA, Complementary; D010940:Plant Proteins; D019895:Transposases