| Selenium is one of the essential trace elements for human body. Selenocysteine, usually residing in the active site of enzyme as catalytic group, is the main form in which the selenium plays in vivo. It has been found that selenocysteine was incorporated co-translationally into the nascent polypeptide. Interestingly, selenocysteine is encoded by codon UGA. Therefore, selenocysteine is also named as the 21st amino acid. It was found that the incorporation of selenocysteine needs a cis-acting element (SECIS) and four proteins (SelA, SelB, SelC, SelD) in E. coli. The bacterial SECIS has a species-specific requirement and it resides immediately downstream of codon UGA encoding selenocysteine in the ORF. Moreover, the incorporation of selenocysteine is a process with greatly low efficiency. Consequently, the research on the expression of heterologous eukaryotic selenoprotein in E. coli is still an area with great challenge. Over the past decades, Enzyme engineering has undergone the most profound and exciting transformation. Rational redesign and directed evolution are two compensatory strategies for enzyme engineering, while rational redesign is more promising for understanding the structure-function relationship and the mechanism by which new enzyme activities evolves from nature. So far, all the examples of enzyme redesign have focused on changing substrate or cofactor specificity or altering the stereochemistry of a reaction, but no body has demonstrated alteration of reaction chemistry. Glutathione S-transferase represents a large enzyme family. It detoxifies endogenous and xenobiotic electrophiles by addition of glutathione to the electrophiles. The distinct structure characteristic, a conserved GSH binding site and a hypervariable hydrophobic substrates binding site, is helpful to understand the structure-function relationship and the evolutional relationship of GST family. Glutathione peroxidase is a well-known selenoenzyme which functions as the antioxidant to protect biomembranes and other cellular components from oxidative damage by catalyzing the reduction of a variety of hydroperoxides, using glutathione as the reducing substrate. Detailed kinetic studies and modeling of enzyme-substrate complexes have suggested that selenocysteines, the catalytic residue in the active site, played critical role in the catalytic cycle because of the specific redox properties of selenium. Both GST and GPX have GSH binding site, only the discrimination between the catalytic residues Tyr (or Ser) and Sec resulted in the divergence in evolution and complete difference in catalytic mechanism. So it is of great interests to investigate whether there is probability to convert GST to GPX by introducing Sec into the active site of GST.1. Construction of selenium-containing GSTOverlap PCR was used for mutagenesis of Tyr7 condon UAU to Sec codon UGA and introducing the minimal SECIS element, which resides downstream of Sec codon UGA in a distance of 11 nucleotides, by substitution for the corresponding nucleotides.To examine whether the bacteria has expressed seleno-GST, protein extracts are analyzed by SDS-PAGE, 75Se-labelled autoradiogram and western-blotting. As shown in the result, no expression band of seleno-GST was detected. Even when it was coexpressed wit genes selA, selB, and selC, there was no detectable band of seleno-GST yet.The Sec incorporation is a process with very low efficiency. Furthermore, the Tyr7 to be substituted by Sec was near the N-terminus. Thus, the incorporation of Sec greatly prolonged the time of initiation step of translation. This resulted in no detectable expression of seleno-GST on the 75Se-labelling autoradiogram. Hence, we inserted the seleno-GST gene into the pGEX-2T plasmid between BamHâ… and EcoRâ… sites.2. Construction of selenium-containing GST fusionIn order to make seleno-GST as fusion protein, we synthesized another pair of primers P3 and P4 to amplify seleno-GST gene. The PCR product was cut with BamHâ… and EcoRâ… and inserted into pGEX-2T plasmid downstrea...
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