An insight into the possible mechanism of working of two-cistronic gene expression systems and rational designing of newer systems.
J Biosci
;
2002 Jun; 27(3): 219-31
Artigo
em Inglês
| IMSEAR
| ID: sea-111222
ABSTRACT
The initial attempts at hyper-expressing buffalo/goat growth hormone (GH)-ORFs in Escherichia 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 an E. 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 in E. 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 under trc promoter) under the T7 promoter in E. 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.
Texto completo:
DisponíveL
Índice:
IMSEAR (Sudeste Asiático)
Assunto principal:
Biossíntese de Proteínas
/
Búfalos
/
Cabras
/
Hormônio do Crescimento
/
Dados de Sequência Molecular
/
Sequência de Bases
/
Engenharia Genética
/
Expressão Gênica
/
Western Blotting
/
Sequência de Aminoácidos
Idioma:
Inglês
Revista:
J Biosci
Ano de publicação:
2002
Tipo de documento:
Artigo
Similares
MEDLINE
...
LILACS
LIS