Quantitative Analysis Of The Read-through Efficiency And Optimization Of The Expression Condition Of Sec In Escherichia Coli

Selenium is an essential element for mammals. Many of selenoenzymes are involved in redox reactions with selenocysteine acting as an essential component of catalytic cycle, such as glutathione peroxidase, thioredoxin reductase, iodothyronine deiodinase and formate dehydrogenase. The expression efficiency of Sec is only 1 -3 % of that of normal amino acids in Escherichia coli, therefore, the selenoprotein is faintly expressed in organism.In prokaryotes, the incorporation of selenocysteine requires four gene products: on tRNA and three proteins. Interestingly, selenocysteine is encoded with UGA codon and incorporated into the polypeptide co-translationally. Since UGA codon remains its routine function in tissues expressing selenoproteins, there is a special regulatory mechanism for the incorporation of selenocysteine. In addition, recognition of UGA as a selenocysteins codon depends on secondary mRNA structures, i.e. stem loops called SECIS (Selenocysteine Inserting Sequence), which locates 11 nucleotides downstream of UGA codon. The SECIS element from prokaryotes is inside the open reading frame of m RNA, while eukaryotic SECIS is in the 5'- or 3'-untranslated region. Furthermore, the bacterial SECIS is species-specific, while the SECIS elements in eubacteria and eukaryotes are somewhat less species-restricted.We had already acquire the expression of GST-Sec in E.coli by the special regulatory mechanism for the incorporation of selenocysteine. To research the influence of selA, selB and selC genes on the read-though efficiency of UGA codon, we use two kinds of report gene system, which are called green fluorescence protein(GFP) reporter andβ-Galactosidase reporter, to detect the read-through efficiency of selenocysteine.GFP reporter system has been constructed by ourselves, then we optimized the condition of expression and detected the read-through efficiency of selenocysteine by the reporter system successfully. The results showed that the read-through efficiency of UGA codon was increased to 1.32-fold in the presence of SECIS element compared to that without SECIS element, and the efficiency was futher increased up to 2.0-fold when GFP fusion was co-expressed with selA, selB and selC genes. These results suggest that GFP can be served as a rapid and convenient tool compared toβ-galactosidase for measuring the read-through efficiency of UGA codon in intact E. coli cells. At the same time, we detected the read-through efficiency of selenocysteine by theβ-Galactosidase reporter system ,too. The results are similar to the GFP.But because of the strong promoter of the vector GFP/pET-32b, the GFP protein will be puckered by mistake, thus the number of GFP molecules will not be in line with the fluorescence intensity of GFP protein, so it makes against to the quantitative analysis of the read-through efficiency of selenocysteine. We have tried to chang vector pUC18 which has weaker promoter to improve the status, but a new problem is that the read-through efficiency of selenocysteine is too low to be detected through the fluorescence apparatus. Perhaps another promoter may improve the matter.Because of the limitation of GFP reporter, we use theβ-Galactosidase reporter system to assess the influence of selA ,selB and selC genes on the read-through efficiency of UGA codon. When co-expressing with selC, the read-through efficiency of Sec was increased by 40% compared to the individual Sec gene. The result is reasonable. However, when co-expressing with the selA or selB gene, the read-through efficiency of UGA codon was decreased by 39% and 89%, respectively. These results suggested that selA and selB did not improve but suppress the read-through of Sec. When co-expressing with the selB and selC gene, the read-through efficiency was increased by 11%. Thus, we proposed that the overexpression of selB protein suppressed the expression level of selA in vivo and decreased the concentration of selenocystl-tRNA^Sec, which resulted in the decrease of read-through efficiency of Sec. In addition, selB may suppress the initiation of translation. At the same time, the read-through efficiency of Sec was enhanced by 5.46 times when Sec gene was co-expressed with the selAB and selC gene. However, the read-through efficiency of Sec was only increased by 2.22 times when Sec was co-expressed with pSUABC under the common conditions accepted by expressing selenoprotein. Obviously our method is better than pSUABC at the read-through efficiency of Sec or selenoprotein expression.