Serine proteases are a family of proteases that utilize an activated serine residue in the sub&not / strate-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. &lsquo / Clp&rsquo / s 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 &ordm / 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 5&acute / end 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. Plasmids of these colonies were analyzed by restriction digestions to select the true recombinants. Expression of the 6xHis-PSP fusion protein from the recombinant E. coli TG-pQE-S1.7 strain was confirmed by functional analysis and SDS-PAGE. An NCBI domain search and multiple sequence alignment using Clustal W 1.82 program indicated homologies between PSP and MSP of Tp. volcanium and various bacterial ATP dependent ClpPs. Signal peptide search using Signal P 3.0 server predicted a signal peptide sequence in MSP homologous to that of Gram (+) bacteria.

In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding intervening sequences is very GC-rich (60.8% G+C) and encodes a preproenzyme of 616 amino acids: a 21 amino acid signal peptide and a 24 amino acid predicted propeptide. lccS gene was amplified using specific primers to exclude the signal and pro-peptide coding regions and ligated to expression vector pAN52-4. The recombinant plasmid was used to transform Aspergillus sojae ATCC11906 (pyrG-). Heterologuos expression was observed in glucose-containing media, under the control of the glyceraldehydes 3-phosphate dehydnogenese promoter and the secretion signal of glucoamylase gene. Laccase gene is an important step towards the high level expression of this enzyme in a GRAS eucaryotic host and for further biotransformation and enzyme engineering studies. In this study also bioinformatic analysis of N-terminal and C-terminal propeptide cleavage sites of fungal proteins including laccases were studied.

国立情報学研究所で電子化したコンテンツを使用している。

The international human rights instruments recognise the inherent dignity and inalienable rights of all members of the human family and that these rights derive from the inherent dignity of the human person. The thesis explores the use of the phrase “inherent dignity” in the context of the catalogue of rights said to be derived from it. (For complete abstract open document)

Thesis (Ph. D.)--Ohio University, August, 2002. / Title from PDF t.p. Includes bibliographical references (leaves 232-247).

Thesis (Ph.D.)--University of Melbourne, Howard Florey Institute of Experimental Physiology and Medicine and Dept. of Anatomy and Cell Biology, 2003. / Typescript (photocopy). Includes bibliographical references (leaves 108-130).

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Surgery, 1999? / Errata is tipped in behind bibliography. Bibliography: leaves 191-215.

Thesis (Ph.D.)--University of Melbourne, Dept. of Philosophy, 2001. / Typescript (photocopy). Includes bibliographical references.