Structure In Translational Initiation Region Research Articles

Expression characteristics of glutamine synthetase of wheat in Escherichia coli

Glutamine synthetase is a key enzyme in plant nitrogen assimilation. To study the structure of wheat glutamine synthetase isoenzymes, GS1, GSr, GSe, GS2 and GS2p of wheat were cloned into pET-21a, and the expression condition was optimized. Although wheat glutamine synthetase isoenzymes had 70%-80% amino acid sequence homology, the isoforms expressed with different characteristics. Induced at 30 °C, the most expression level of GSr, GSe and GS2 was after 3 h, and of GS1 was at the 7 h whereas no GS2p was expressed, and the GS isoenzymes showed different expression level, with the order of GS1 (22%)>GSr (15%)>GS2 (12%)>GSe (5%). GSe expressed as soluble protein, and GS1 expressed mainly as soluble protein whereas GSr and GS2 expressed as insoluble proteins. Induced at 30 °C for 3 h, mRNA transcript levels of GS isoforms were different, with the order of GSr (7.59)>GS2 (1.84)>GS2p (1.66)>GSe (1.46)>GS1 (1.00). The levels of mRNA transcription were not consistent with the level of the protein translation. The analysis of mRNA secondary structure showed the free energy of translation initiation region of glutamine synthetase isoforms was different, with the order of GS1 (14.4)<GSr (17.2)<GS2 (22.6) <GSe (25.4) <GS2p (31.6), the smaller freed energy, the more unstable mRNA secondary structure of translation initiation region and the higher level of protein expression. Soluble expression condition of glutamine synthetase isozymes was also different, with GS1, GSr, GSe and GS2 induced at 30 °C for 5 h, 16 °C for 15 h, 37 °C for 5 h, and 25 °C for 7 h respectively. The soluble protein showed different expression level with GS1 (20%)>GSr (13%)>GS2 (10%)>GSe (7%), and different activities with GS1>GSe>GS2, and the activity of GSr was not detected. The gene sequence of glutamine synthetase isoenzymes determines the amount, status and activity of proteins expressed in prokaryotic cells.

Optimizing the expression of &lt;I&gt;Mx&lt;/I&gt; gene in &lt;I&gt;Escherichia coli&lt;/I&gt; based on rare codon and mRNA structure

Rare codon and mRNA secondary structure of translation initiation region were analyzed. Four bioengineered bacterium BL21(DE3)/pET-Mx, Rosseta (DE3)/pET-Mx, BL21(DE3)/pGEX-Mx, and Rosseta (DE3)/pGEX-Mx were obtained. Through optimizing the rare codon and mRNA secondary structure of translation initiation region, Mx protein was expressed in Rosseta (DE3)/pET-Mx, and Rosseta (DE3)/pGEX-Mx. The specified product of 75 kDa was detected by means of Western blotting analysis. The result showed that Rosetta (DE3) strain expressing some rare codon used in our experiment can obtain Mx protein expression, and the lower energy of mRNA structure can improve the expression level of Mx protein. This is the first report on the expression of the full open reading frame of Mx gene.

Overexpression of GH10 endoxylanase XynB from Thermotoga maritima in Escherichia coli by a novel vector with potential for industrial application

The GH10 endoxylanase B encoded by xynB of Thermotoga maritima is a particularly attractive candidate for some industrial purposes. In present study, we aimed to develop an expression system to increase the enzyme production and decrease the costs in the induction of xynB expression in Escherichia coli. The xylanase was overexpressed in E. coli by using the novel pHsh expression vector. After optimization of the mRNA secondary structure in translational initiation region of pHsh- xynIII, the Gibbs free energy of 73 nt was changed from −10.5 kcal mol −1 to −6.71 kcal mol −1, and the SD sequence was completely released to a large loop. The nucleotide sequence changes resulted in the increase of expression level of xynB from 276 U ml −1 to 489 U ml −1 in LB medium, and from 977 U ml −1 to 1893 U ml −1 in Terrific broth. Moreover, the recombinant XynB was successfully expressed in fermentors by using “flow-in-heat” method. Thus, this report provides an industrial means to produce the extremely thermostable xylanase in E. coli.

Temperature influences on the expression of GFP promoted by the upstream sequence of cpcB from Arthrospira platensis

In order to investigate the regulation mechanism of the phycocyanin gene, a series of functional analyses of the upstream sequence of cpcB gene from Arthrospira platensis were conducted in E. coli with green fluorescent protein encoding gene (gfp) as the reporter. Results showed that the gfp gene could express at a high level under the promotion of the upstream sequence, suggesting the existence of some strong promoter elements in it. The expression of GFP was influenced by temperature. Higher temperature led to higher expression level. The bioinformatics analyses followed by mutation analyses on the secondary structure of translation initiation region (TIR) revealed that RNA thermosensor might account for the temperature regulation.