Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli.
We have designed a novel coupled transcriptional construct wherein Escherichia coli uracil DNA glycosylase (UDG) and Bacillus subtilis phage PBS-2 encoded uracil DNA glycosylase inhibitor protein (Ugi) genes were cloned in tandem, downstream of an inducible promoter (Ptrc). Use of this bicistronic operon has allowed purification of large amounts of UDG-Ugi complex formed in vivo. The system has also been exploited for purification of large amounts of Ugi. While establishing the expression system, one of the constructs showed detectable suppression of UAG termination codon and resulted in accumulation of a minor population of a putative readthrough polypeptide corresponding to UDG. We discuss the likely occurrence of such a phenomenon in overproduction of other recombinant proteins. Finally, the usefulness of the operon construct in convenient mutational analysis to study the mechanism of UDG-Ugi interaction is also discussed.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-, 560 012, India.
S
S
Roy
K
K
Purnapatre
P
P
Handa
M
M
Boyanapalli
U
U
Varshney
D017103:Bacillus Phages; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D045647:DNA Glycosylases; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000235:genetics; D009876:Operon; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014158:Transcription, Genetic; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase