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1	Expression Of Recombinant Acid Protease (thermopsin) Gene From Thermoplasma Volcanium Koyuncu, Bilsev 01 January 2006 (has links) (PDF) 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. QH Genetics 426-470
2	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760
3	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760

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