Data/hora: 01/06/2024 11:21:59

Provedor de dados: | 74
País: | Brazil
Título: | Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion
Autores: | Wang,Zhongshan; Xiang,Quanju; Wang,Guangjun; Wang,Haiyan; Zhang,Yizheng.
Data: | 2011-01-01
Ano: | 2011
Palavras-chave: | Escherichia coli; Glutathione transporter; GsiA; Gene expression; Green fluorescent protein.
Resumo: | The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.
Tipo: | Info:eu-repo/semantics/article
Idioma: | Inglês
Identificador: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572011000400019
Editor: | Sociedade Brasileira de Genética
Relação: | 10.1590/S1415-47572011005000043
Formato: | text/html
Fonte: | Genetics and Molecular Biology  v.34 n.4 2011
Fonte: | info:eu-repo/semantics/openAccess