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1	Extracellular Recombinant Human Growth Hormone Production By Pichia Pastoris Orman, Mehmet Ali 01 August 2007 (has links) (PDF) In this study, the effects of bioprocess operation parameters on recombinant human growth hormone (rhGH) production by P. pastoris were systematically investigated. In this frame, first, for the extracellular expression and purification of human growth hormone by recombinant P. pastoris the cDNA of hGH, fused with a polyhistidine tag and also fused with a target site for the Factor Xa protease in which cleavage produces a mature N- and C- termini of rhGH, was cloned into pPICZ&amp / #945 / A plasmid and the constructed system within the plasmid, pPICZ&amp / #945 / A::hGH, was integrated to AOX1 locus of P. pastoris and expressed under alcohol oxidase promoter which is induced by methanol. With dot-blot analysis, the appropriate two strains producing human growth hormone at high levels and having different methanol utilization phenotype (Mut+ and Muts) were chosen among the other transformants. Then, the effects of methanol concentrations on the expression of rhGH and cell growth were analyzed and both of the phenotypes were compared in defined and complex media in laboratory scale air filtered shake bioreactors. The highest rhGH concentration for Mut+ and MutS, was found as 0.052 kg m-3 and 0.16 kg m-3, respectively, at 2 %(v/v) methanol concentration in complex medium. When methanol was used as the sole carbon source in defined medium, Muts phenotype had very low specific growth rate on methanol due to the intrinsic characteristics of it, therefore detectable rhGH was not observed, on the other hand, optimum rhGH concentration produced by Mut+ strain was found as 0.032 kg m-3 at 3% (v/v) methanol concentration in defined medium. In mixed system (glycerol/methanol) which is also defined, when the optimum glycerol concentration, 30 kg m-3, was used, Muts produced the highest rhGH, 0.110 kg m-3, at 1% (v/v) methanol concentration and any increase in methanol concentration resulted in lower rhGH production, on the other hand, Mut+ strain produced 0.060 kg m-3 rhGH at 4% (v/v) methanol concentration, which indicated that higher rhGH production capacity of Mut+ strain was obtained at high methanol concentrations. Using the designed defined medium for Mut+ phenotype where methanol was used as the sole carbon source with an optimum concentration of 3% (v/v), the effects of oxygen transfer on rhGH production, by-product formation, and cell growth, oxygen transfer and fermentation characteristics were investigated by using pilot scale bioreactor. Oxygen transfer effects on rhGH production were investigated at QO/VR=0.5 vvm / N=250, 500, 625, 750 min-1 conditions. The variations in rhGH , cell, amino acid and organic acid concentrations with the cell cultivation time, specific cell growth rate, the oxygen uptake rate, the liquid phase coefficient by using the dynamic method, maintenance coefficient for oxygen and yield coefficients were determined. The highest rhGH concentration was obtained at 0.5 vvm, 500 min-1 condition as 0.023 kg m-3 with 5.37 kg m-3 cell density. QR Microbiology 1-502
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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