An insight into the possible mechanism of working of two-cistronic gene expression systems and rational designing of newer systems

Kumar, Utpal ; Sahni, Girish (2002) An insight into the possible mechanism of working of two-cistronic gene expression systems and rational designing of newer systems Journal of Biosciences, 27 (3). pp. 219-231. ISSN 0250-5991

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Official URL: http://www.ias.ac.in/jbiosci/jun2002/219.pdf

Related URL: http://dx.doi.org/10.1007/BF02704911

Abstract

The initial attempts at hyper-expressing buffalo/goat growth hormone (GH)-ORFs inEscherichia coli directly under various strong promoters were not successful despite the presence of a functional gene. High level expression of GH was achieved as a fusion protein with glutathione-S-transferase (GST). To produce native GH in an unfused state, we adapted an established strategy of two-cistronic approach in our system. In this strategy, utilizing one of the highly efficient reported sequences as the first cistron led to a nearly 1000-fold enhancement in the level of expression under anE. coli promoter (trc). In search of a newer first-cistron sequence as well as to see the generality of the two-cistronic approach, we explored the ability of different lengths of a highly expressing natural gene to act as an efficient first cistron. Surprisingly,GST, which is naturally highly expressible inE. coli, could not be fitted into a successful two-cistronic construct. In addition, placement of the entire two-cistronic expression cassette (which had earlier given high-level GH expression undertrc promoter) under theT7 promoter inE. coli failed to hyper-express GH. These results suggest that the successful exploitation of the two-cistron arrangement for hyper-expression of eukaryotic ORFs in bacteria is not as straightforward as was previously thought. It appears probable that factors such as the sequence context, together with the length and codons used in the first cistron are important as well.

Item Type:Article
Source:Copyright of this article belongs to Indian Academy of Sciences.
Keywords:Escherichia coli; Gene Expression; Growth Hormone; mRNA Secondary Structure; Translational De-repression; Two-Cistron
ID Code:45237
Deposited On:27 Jun 2011 04:58
Last Modified:18 May 2016 01:34

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