Cloning and expression of periplasmic (clp p-like) and membrane-bound serine protease genes of thermoplasma volcanium in escherichia coli

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2006

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Demirok, Burçak

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Serine proteases are a family of proteases that utilize an activated serine residue in the substrate-binding pocket to catalytically hydrolyze peptide bonds. Enzymes which belong to this family, with a diverse array of metabolic and regulatory functions, play critical roles in cell physiology and pathology. ءClp̕s are a class of ATP dependent serine proteases which are composed of a protease (ClpP) and an ATPase (ClpA or ClpX) component. Their involvements in degrading proteins are especially implicated under stress conditions. In contrast to members of Bacteria and Eukarya, little is known about the energy-dependent proteolysis and there is no report on Clp family proteases in Archaea. In this study, for the fist time, a periplasmic Clp P-like (PSP) and a membrane bound serine protease (MSP) genes from thermophilic archaeon Thermoplasma volcanium GSS1 were cloned and expressed in E. coli. PCR amplifications at 55 ð C yielded unique fragments of 971 and 1521bp, for PSP and MSP genes, respectively, which were ligated to p-Drive cloning vectors and introduced into E.coli TG1 competent cells. Recombinant clones were screened depending on blue/white colony selection. Putative recombinant plasmids were analyzed by restriction enzyme digestions. Serine protease activities of the three positive clones (E. coli TG-S1, E. coli TG-S4 and E. coli TG-M1) were determined spectrophotometrically by using chromogenic oligopeptide substrates. These results indicated that cloned PSP and MSP genes were successfully expressed in E. coli under the control of their own promoters. Heterologous expression of PSP gene was also attempted by adding 6xHis tag to the 5énd of the PSP gene in pQE 30 expression vector. Competent E.coli TG1 cells were transformed by pQE expression constructs. Positive clones were detected on colony blots using Anti-His HRP conjugates and chromogenic DAB substrate.

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Genetics.

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http://etd.lib.metu.edu.tr/upload/3/12606994/index.pdf
https://hdl.handle.net/11511/15652

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

B. Demirok, “Cloning and expression of periplasmic (clp p-like) and membrane-bound serine protease genes of thermoplasma volcanium in escherichia coli,” M.S. - Master of Science, Middle East Technical University, 2006.