| Glutathione peroxidase (GPX) is a member of the antioxidant enzyme system, which plays an important role in protection of organisms. GPX distribute extensively in cell, blood, and tissues. GPX prevent the organism from the damage of free radicals by scavenging hydroxide and lipid hydroperoxide. It was demonstrated that GPX activity decreased when organism suffered from some disease such as diabetes, Keshan disease and cataract etc, therefore, GPX is a powerful candidate for the antioxidant medicine. Due to the disadvantages of native GPX, such as instability and limited availability, great efforts have been made in the artificial mimic of GPX.The catalytic residue of GPX is selenocysteine (Sec) which is encoded by an opal codon UGA, usually as a stop codon in all organisms investigated so far. The elucidation of how Sec is incorporated into protein has progressed rapidly in both prokaryotic and eukaryotic systems. In Escherichia coli, there are a cis-acting element SECIS (selenocysteine insertion sequence) and four trans-acting factors, including SelA, SelB, SelC and SelD, involved in the selenocysteine incorporation. SelC encodes a selenocysteine-specific tRNA (seryl-tRNASec) which can be converted to selenocysteinyl-tRNASec by the action of selenocysteine synthetase (SelA). The active selenide is donated by selenophosphate which is synthesized from selenide and ATP by selenophosphate synthetase (SelD). SelB is a specific elongation factor that can interact specifically with both tRNASec and SECIS. Then the quaternary complex SelB?GTP?Sec-tRNASec?SECIS is formed and selenocysteine is inserted into the selenopolypeptide at the site containing UGA codon. In prokaryotes, SECIS elements locate immediately downstream of UGA, whereas in eukaryotes SECIS elements locate in the 3' untranslated regions of the selenoprotein-coding mRNA. Moreover, the bacterial SECIS was species-specific while SECIS in eubacteria and eukaryotes has no species-specific requirement. The SECIS of fdhF, encoding 80kD subunit of formate dehydrogenase H in E. coli, has been demonstrated in detail. The minimal SECIS, an upper stem-loop structure of SECIS, consists of 17 nucleotides and resides in s distance (11 nucleotides) from the UGA codon.Our antibodies with glutathione binding sites, which were chemically modified, exhibited high GPX activity. But the method of chemically modifying proteins has many shortcomings. So we wanted to express the scFv2F3 containing Sec in E. coli with selenoprotein synthesis system of E. coli. We mutated Ser52 to Sec52 in CDR2 of heavy chain. The minimal fdhF SECIS consisting of 17 nucleotides, was introduced into the gene of scFv2F3 positioning 11 nucleotides downstream of Sec52 codon UGA by mutating the corresponding nucleotides. To exam the expression of scFv2F3 containing Sec, we amplified four genes:The gene of scFv2F3 without mutation, named F, as control;The mutant gene of scFv2F3 with Sec codon, named FT, as control;The mutant gene of scFv2F3 with minimal SECIS, named FS, as control;The mutant gene of scFv2F3 with both Sec codon and minimal SECIS, named FTS, aim gene;The four genes were recombined with vector pET21a(+) and the recombined plasmids were expressed in BL21(DE3) which had been transformed plasmid pSU which can improve the synthesis of selenoprotein. The result of Se75-labeling showed that the scFv2F3 containing Sec was expressed in E. coli with the help of pSU, but the expression level was too low to be seen in SDS-PAGE. We wanted to find the aim protein in the washed inclusion body, but we failed. Then we changed the expression system to fusion expression system to improve the expression level of aim protein. Perhaps we would obtain soluble aim protein which can be purified with glutathione 4B-Sepharose. The result of Se75-labeling showed that the fusion protein of scFv2F3 containing Sec with GST was expressed in E. coli with or without the help of pSU, but the expression level was still very low so that the protein was difficult to be seen in SDS-PAGE. Mo...
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