Roy, Sudipta ; Purnapatre, Kedar ; Handa, Priya ; Boyanapalli, Madanamohan ; Varshney, Umesh (1998) Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli Protein Expression and Purification, 13 (2). pp. 155-162. ISSN 1046-5928
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Official URL: http://www.sciencedirect.com/science/article/pii/S...
Related URL: http://dx.doi.org/10.1006/prep.1998.0878
Abstract
We have designed a novel coupled transcriptional construct whereinEscherichia coliuracil DNA glycosylase (UDG) andBacillus subtilisphage 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 formedin 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.
| Item Type: | Article |
|---|---|
| Source: | Copyright of this article belongs to Elsevier Science. |
| Keywords: | Coupled Transcription; Bicistronic mRNA; Termination Codon; Ung |
| ID Code: | 56227 |
| Deposited On: | 23 Aug 2011 11:51 |
| Last Modified: | 23 Aug 2011 11:51 |
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