Energy generating processes like the citric acid cycle are a pivotal part of metabolism. Members of the 2-oxoacid dehydrogenase multienzyme complex (OADHC) superfamily feed into and act within the citric acid cycle. OADHCs are composed of three enzymes: 2-oxoacid decarboxylase (E1), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). Covalent attachment of lipoic acid (LA) to E2 is essential for overall OADHC activity. Although thought to be absent in Archaea, it has recently been found that Thermoplasma acidophilum has all the components for an active recombinant OADHC (Heath et al., 2007). Recent studies have further suggested that Tp. acidophilum may have an enzyme to covalently attach LA to E2 (Sun et al., 2007; McManus et al., 2006). This work describes the cloning and recombinant expression of the Thermoplasma lipoate protein ligase (Tp. LplA), its C-terminal domain and a fusion protein composed of the above two proteins. Both proteins are required for lipoylation of E2 in vitro. For the first time, in vivo lipoylation of E2 in Tp. acidophilum cell cultures is also being reported. The effect of lipoylation and temperature on the Thermoplasma OADHC assembly has also been studied. This study revealed the temperature dependence of the E2 core and the whole complex assembly. These findings are in line with the optimum growth temperature of Tp. acidophilum. Dynamic light scattering and analytical ultracentrifugation were used to determine the molecular mass of whole OADHC. The molecular mass was determined to be 5 MDa with an octahedral geometry of the E2 core. The results of this work strengthen the assumption that these enzyme systems may have had or potentially have a role in the Archaea. This may hold further clues to the evolutionary relationship between the three kingdoms of life and the role of OADHCs/lipoylation in the Archaea. The temperature dependent assembly of the complex and thermostability of these proteins may also provide a model to study thermostability and protein-protein interactions at high temperatures.

Universiẗat, Diss., 2002--München.

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.

Techn. Universiẗat, Diss., 2003--München.

Techn. Universiẗat, Diss., 2003--München.

No description available.

Chaperonins (Hsp60 chaperones) comprise a class of oligomeric, high-molecular-weight chaperones that have the unique ability to fold some proteins that cannot be folded by simpler chaperone systems. The term &ldquo / thermosome&rdquo / is used for molecular chaperonins from Archaeal organisms since they accumulate to high levels upon heat-shock. In this study first time, we have cloned and sequenced two Hsp60 subunit genes (&amp / #945 / and &amp / #946 / ) from a thermoacidophilic archaeon Thermoplasma volcanium. For cloning we have followed a PCR based strategy. Amplification of Hsp60 &amp / #945 / gene from chromosomal DNA of T. volcanium yielded a product of 1939 bp amplicon and that of Hsp60 &amp / #946 / gene yielded a product of 1921 bp amplicon. After ligation of the PCR fragments to pDrive vector, recombinant plasmids were transferred into E. coli TG-1 competent cells and recombinant colonies were selected by blue/white screening. The cloning of two subunit genes were confirmed by restriction mapping and by sequencing. Both subunit genes were then subcloned to pUC18 vector consequtively to construct a co-expression vector. Both subunit genes were expressed under control of their own promoters leading to production of active Hsp60 chaperonin (thermosome). Chaperone activity of the recombinant thermosome was shown by using pig citrate synthase enzyme as substrate. Thermosome induced refolding was observed when renaturation was carried out at 50&deg / C for 2,5 h. Under this condition, citrate synthase activities associated with control and test were &amp / #61508 / mA412/min:19.0 and &amp / #61508 / mA412/min:24.0 respectively. Clustal W Version 1.82 was used for multiple sequence alignments of Hsp60 &amp / #61537 / and Hsp60 &amp / #61538 / proteins of T. volcanium and other Hsp60 proteins from various eukaryotes, bacteria and archaea. The highest sequence similarity was found between &amp / #945 / subunit proteins of T. volcanium and T. acidophilum (94%) and &amp / #946 / subunit proteins of T. volcanium and T. acidophilum (93%). Clusters of orthologous groups and conserved domain database searches revealed the phylogenetic relationships between Hsp60 &amp / #61537 / and Hsp60 &amp / #61538 / subunits of T. volcanium thermosome and other Hsp60 proteins from various eukaryotes, bacteria and archaea. Induction of both subunit genes under heat shock (65&deg / C, 70&deg / C and 75&deg / C for 2h) and under oxidative stress (imposed by 0,008 mM, 0,01 mM, 0,02 mM, 0,03 mM and 0,05 mM H2O2) conditions was studied by Real-Time PCR technique and amplified cDNA band density analysis.

In this study, possible involvement of the small heat shock proteins (sHsps) from a thermoacidophilic archaeon, Thermoplasma (Tp) volcanium in the stress response was investigated. Our results showed that heterologous, high level expression of TVN0775/sHsp gene in E.coli increased its thermotolerance at 53&deg / C for two hours. But, the second sHsp of the Tp. volcanium, TVN0984/sHsp was not effective in improvement of the thermal resistance of the mesophilic bacterium (i. e., E.coli). The expression of the TVN0775/sHsp and TVN0984/sHsp genes increased about 3 fold after heat-shock at 65&deg / C, as revealed by Real-Time PCR analysis. Although expression of the both genes was induced at 70&deg / C, TVN0984/sHsp gene expression was increased higher (about 5 fold) than that of the TVN0775/sHsp gene expression (about 1.5 fold). Tp. volcanium cells were exposed to high pH (pH: 3.5, pH: 4.0, pH: 4.5, pH: 5.0), and the change in the sHsp genes&rsquo / expression profile were analyzed. The results showed that TVN0775/sHsp gene expression was more sensitive to increased pH than TVN0984/sHsp gene expression. The TVN0775/sHsp gene transcription induced at most 2.5 fold at pH 4.0 and the gene expression either reduced or did not change at higher pH values (i.e., pH 4.5 and 5.0). On the other hand, TVN0984/sHsp gene expression did not change at pH 4.0 but significantly reduced at higher pH values. The effect of oxidative stress on the expression of TVN0775 and TVN0984 genes was investigated by treatment of Tp. volcanium cells with 0.01 mM, 0.02 mM, 0,03 mM and 0.05 mM H2O2. For both sHsp genes, transcription was induced at lower concentrations of H2O2 (0.01 mM and 0.02 mM). At higher concentrations of H2O2 expression of both genes&rsquo / transcription either did not changed or down regulated. Lastly, in this study we have purified the recombinant TVN0775/sHsp, as an Nterminal 6x his-tag fusion to homogeneity on Ni-NTA affinity column. Purified protein samples were used in the chaperone activity assays using bovine glutamate dehydrogenase enzyme (boGDH) as substrate. We have found that the recovery of glutamate dehydrogenase activity at 45&deg / C, 50&deg / C and 53&deg / C in the presence of the Tp. volcanium sHsps was higher than that of spontaneous refolding. Also, TVN0775/sHsp increased the recovery of the boGDH enzyme that was denatured at 2.5 M GdnHCl concentrations for 30 min.

In this study first time, we have cloned and sequenced the citrate synthase gene from a thermoacidophilic archaeon Thermoplasma (Tp.) volcanium (Optimum growth temperature of Tp.volcanium is 60oC and optimum pH is 2.0.). For cloning we have followed a PCR based approach. Amplification of citrate synthase gene from chromosomal DNA of Tp.volcanium yielded a product of 1476 bp containing an open reading frame of 1161 bp comprising the structural gene. After ligation of the PCR amplicon to pDrive vector through AU complementation, recombinant plasmids were transferred into E.coli TG-1 competent cells. Out of three recombinants, E.coli pDriveCS-31 was selected for further characterization by restriction mapping and DNA sequencing. Southern Blotting and Hybridization using the membrane blot of pDriveCS-31 plasmid and DIG-labeled PCR amplified citrate synthase gene probe, also confirmed the cloning of Tp.volcanium citrate synthase gene in E.coli. Clustal W Version 1.82 was used for alignment of aminoacid sequence of Tp.volcanium citrate synthase with that of other archaeal, bacterial and eukaryotic citrate synthases. The highest sequence similarity (87%) was found between Tp.volcanium and Tp.acidophilum enzymes. Despite low sequence homology (18%) with the pig enzyme, of the 11 residues implicated in catalytic activity of the pig citrate synthase 9 were conserved in the Tp.volcanium enzyme. Heterologous expression of this citrate synthase gene in E.coli has been achieved under the control of its promoter sequences. The recombinant enzyme (386 aa) has been purified to homogeneity by affinity chromatography on Reactive Red 120 column. The subunit molecular size was estimated as 43 kDa. The purified enzyme followed classical Michaelis-Menten kinetics. The Km values of 5.15 &amp / #956 / M and 5.60 &amp / #956 / M, and Vmax values of 1.74 &amp / #956 / moles/ml/min and 1.60 &amp / #956 / moles/ml/min were calculated from Lineweaver-Burk plots for acetyl-CoA and oxaloacetate, respectively. The recombinant enzyme was thermostable and retained about 80% of the activity at 85oC after 1 hour.

Acid proteases, commonly known as aspartic proteases are degredative enzymes which catalyze the cleavage reaction of peptide bonds in proteins with a pH optimum in the acidic range (pH 3-4). Acid proteases have crucial roles in metabolism. Moreover, they are used in different fields of industry. Thermophilic microorganisms, especially archaea, gain special interest because of their thermal stability for both fundamental and industrial researches. Thermopsin is an extracellular acid protease and a member of A5 family of proteases. This thermophilic enzyme has no characteristic active aspartyl residue, is insensitive to pepstatin and no apparent sequence homology to other acid proteases and therefore represents a new class of acid proteases. Thermophilic archaeal strain Thermoplasma volcanium GSS1 (optimum temperature 550C and pH 2.7) in the genome has a putative thermopsin gene encoding 998 amino acid enzyme. In this study thermopsin gene from Thermoplasma volcanium was expressed in E. coli as fusion with 6xHis tag under the control of T5 transcription/translation system. Putative thermopsin gene from Thermoplasma volcanium was amplified by PCR method using two primer sets and cloned. A 3080 bp and a 3070bp PCR products were obtained by using TP1/TP2 primer set (thermopsin gene with the start codon) and TP1&rsquo / /TP2 primer set (thermopsin gene missing start codon) respectively. PCR amplified thermopsin genes pDrive and pUC18 vectors in E. coli TG1 were cloned using and then cloned genes were sub-cloned directionally into pQE triple vector set for expression. In these expression vectors, cloned genes are placed downstream of a 6XHis tag to produce an expression fusion. E.coli strains (M15[pREP4], SG13009[pREP4], and TG1) used as hosts. Recombinant colonies screened by colony blot/hybridization method based on immunological detection of the expressed 6XHis tag fusion by Anti-His HRP conjugates which are specific for 6xHis tag, and DAB chromogenic substrate was used for colony blot procedure. PCR amplified thermopsin gene containing 3080bp could not expressed in pQE30 and 31 vectors in TG1 strains. It is thought that pQE32 open reading frame can be true for thermopsin gene (3080bp). Three expression constructs, pQE31-1, pQE31-4 and pQE31-6 plasmids containing PCR amplified 3070bp thermopsin gene were confirmed as true recombinant plasmids according to both colony blot hybridization result and restriction digestion profile the agarose gel.