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Articles 1 - 12 of 12

Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Protein-Carbohydrate And Protein-Protein Interactions: Using Models To Better Understand And Predict Specific Molecular Recognition, Alain Teboho Laederach Jan 2003

Protein-Carbohydrate And Protein-Protein Interactions: Using Models To Better Understand And Predict Specific Molecular Recognition, Alain Teboho Laederach

Retrospective Theses and Dissertations

Any molecular recognition event results in a change in the free energy of the system. The extent of this change is related to the association constant, such that the more negative the free energy change is, the tighter the interaction between receptor and ligand. Protein-carbohydrate interactions play a critical role in signal transduction, innate immunity, and metabolism. Modeling these interactions is somewhat complicated by the inherent flexibility of carbohydrates as well as their relatively large number of functional groups. An empirical scoring function for docking carbohydrates to proteins, specifically tailored to predict both the correct binding orientation and free energy ...


Recovery Of Recombinant Proteins From Transgenic Plants: Strategies In Implementation And Optimization , Todd J. Menkhaus Jan 2002

Recovery Of Recombinant Proteins From Transgenic Plants: Strategies In Implementation And Optimization , Todd J. Menkhaus

Retrospective Theses and Dissertations

Strategies to optimize the recovery of recombinant proteins from plant hosts have been investigated. The use of different crops, different product molecules, and different separation techniques was studied to suggest modes of operation that reduce the overall processing cost for obtaining purified recombinant proteins from plants.;Polyelectrolyte precipitation of beta-glucuronidase (GUS), an acidic model protein, with polyethyleneimine (PEI), a polycatonic polymer, from the extracts of canola, corn germ, soybean, and pea met with varying degrees of success. Precipitation from canola required the least amount of PEI and was capable of providing the highest enrichment. In contrast, soy and pea allowed ...


Investigation Of The Effect Of Bcl-2 On Hybridoma Cell Survival , Mark Christopher Mowry Jan 2001

Investigation Of The Effect Of Bcl-2 On Hybridoma Cell Survival , Mark Christopher Mowry

Retrospective Theses and Dissertations

Hybridoma cells are widely used to produce large quantities of monoclonal antibodies for use as medicines and diagnostics. Bcl-2 is an oncogene that has been shown to have an anti-apoptotic effect in many cell lines including hybridomas. This study investigates the effect of various levels of bcl-2 expression on hybridoma cell grown and antibody productivity in both batch and perfusion culture.;H166 hybridomas were transfected with a plasmid containing a bcl-2 insert and stable clones were selected. The cells were grown in spinner flasks in batch mode for twelve days and it was confirmed that expression at high levels allow ...


Enhancing Chromatographic Separations Of Recombinant Proteins From Canola Extracts By Genetic Design And Characterization Of Protein Binding Regions , Chenming Zhang Jan 1999

Enhancing Chromatographic Separations Of Recombinant Proteins From Canola Extracts By Genetic Design And Characterization Of Protein Binding Regions , Chenming Zhang

Retrospective Theses and Dissertations

Canola was studied as an alternative source to traditional microbial systems as a recombinant protein production host. Various chromatographic methods were used in this work to systematically characterize the native canola protein elution profiles, and different genetically engineered proteins were selected to explore the opportunities presented for effective protein recovery;Native canola protein was eluted into two major peaks in linear salt gradient elution on cation-exchange chromatography. T4 lysozyme and its mutants, by both point mutation and fusion, were used as model proteins to investigate the charge effect on protein elution. It was found that the single mutant T4 lysozyme ...


Mutational Analysis Of Aspergillus Awamori Glucoamylase Selectivity To Improve Glucose Yield , Hsuan-Liang Liu Jan 1998

Mutational Analysis Of Aspergillus Awamori Glucoamylase Selectivity To Improve Glucose Yield , Hsuan-Liang Liu

Retrospective Theses and Dissertations

Three single mutations, Lys108→Arg, Lys108→Met and Tyr312→Trp, and one insertional mutation, 322-314 Loop, in Aspergillus awamori glucoamylase were constructed and tested along with four previously constructed thermostable mutations: Asn20→Cys/Ala27→Cys (SS), Ser30→Pro, Gly137→Ala, and Ser436→Pro, and one previously made thermosensitive mutation, Ala27→Pro, for their selectivity by high-concentration glucose condensation and maltodextrin hydrolysis reactions. Lys108 is important in substrate binding during maltooligosaccharide hydrolysis. Both SS and 311-314 Loop mutant GAs successfully decrease isomaltose formation rate and thus increase peak glucose yield. Tyr312→Trp GA helps to form a tighter hydrogen bond network ...


Computational Studies Of Glucoamylase Selectivity , Pedro Maldonado Coutinho Jan 1996

Computational Studies Of Glucoamylase Selectivity , Pedro Maldonado Coutinho

Retrospective Theses and Dissertations

Glucoamylase (GA) is an industrial enzyme involved in the production of glucose and fructose syrups from starch. Exhibiting a large spectrum of selectivities, GAs are able to cleave glucose from the nonreducing ends of [alpha]-(1,4) glycosidic bonds of maltooligosaccharide chains and the [alpha]-(1,6) bonds initiating their branches. The high temperatures and glucose concentrations occurring in saccharification lead to the formation of condensation products, reducing the overall glucose yield. Modified versions of Aspergillus GA that better satisfy industrial requirements are desirable. To achieve this, a structure-based multisequence alignment of the primary sequences of the catalytic domains, linkers ...


Site-Directed Mutagenesis To Enhance Thermostability Of Aspergillus Awamori Glucoamylase Expressed In Saccharomyces Cerevisiae , Hsiu-Mei Chen Jan 1993

Site-Directed Mutagenesis To Enhance Thermostability Of Aspergillus Awamori Glucoamylase Expressed In Saccharomyces Cerevisiae , Hsiu-Mei Chen

Retrospective Theses and Dissertations

A program of site-directed mutagenesis was undertaken to enhance thermostability of Aspergillus awamori glucoamylase. Nine mutations were designed, based on three different irreversible thermoinactivation mechanisms of glucoamylase. Each mutant was expressed in Saccharomyces cerevisiae, and their production, activities, and irreversible thermoinactivation rates were determined;First, the Asn182 and Asn395 residues were each replaced with Gln to reduce their deamidation rates. Asn395, but not Asn182, was N-glycosylated by yeast. Deletion of the N-glycan linked to Asn395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. Replacement of Asn182 by Gln did not change glucoamylase specific activity or thermostability ...


Enhancement Of Polyelectrolyte Precipitation Through The Genetic Fusion Of Charged Polypeptides To Enzymes , Mark Quinn Niederauer Jan 1993

Enhancement Of Polyelectrolyte Precipitation Through The Genetic Fusion Of Charged Polypeptides To Enzymes , Mark Quinn Niederauer

Retrospective Theses and Dissertations

The focus of this work was to investigate the enhancement of polyelectrolyte precipitation through the genetic alteration of the target protein. The dissertation is divided into three primary chapters: a literature review of precipitation techniques which display high selectivity, experimental work including the characterization and precipitation of the fusion enzymes, and modeling of the precipitation process;The literature review of selective precipitation techniques focuses on those techniques for which relatively high purifications can be achieved. The techniques include precipitation with polyelectrolytes, affinity ligands, metal ions, and protein-binding dyes. Topics discussed include the mechanisms of selectivity and precipitation, degrees of purification ...


Tailoring The Ph Dependence Of Glucoamylase From Aspergillus Awamori By Mutagenesis , Ufuk Bakir Jan 1993

Tailoring The Ph Dependence Of Glucoamylase From Aspergillus Awamori By Mutagenesis , Ufuk Bakir

Retrospective Theses and Dissertations

A program of cassette mutagenesis was undertaken in the active site of Aspergillus awamori glucoamylase, mainly to alter the effect of pH dependence of the enzyme but also to determine possible functions of the mutated residues. For this purpose, nine single amino acid mutants were prepared and expressed in Saccharomyces cerevisiae, and their effects were determined by using steady-state enzyme kinetics;Three different groups of mutations were designed. In the first group, Glu179 → Asp, Asp176 → Glu, and Glu180 → Asp mutations were prepared. The Glu179 → Asp mutation led to a very large decrease in k[subscript] cat but no change in ...


Characterization Of Substrate Binding And Catalytic Mechanisms Of An Endoxylanase, Amylosucrase, And Porcine Pancreatic Alpha-Amylase , Bernard Yi Tao Jan 1988

Characterization Of Substrate Binding And Catalytic Mechanisms Of An Endoxylanase, Amylosucrase, And Porcine Pancreatic Alpha-Amylase , Bernard Yi Tao

Retrospective Theses and Dissertations

The subsite affinity map of an endo-(1 → 4)-[beta]-xylanase produced by Aspergillus niger has been determined by using linear, [superscript]3H-reducing-end-labeled xylooligosaccharides ranging in length from xylotriose through xylooctaose. No evidence of multiple attack or of condensation and transxylosylation reactions was found. Bond cleavage frequencies were highest near the reducing end of short substrates, with the locus of highest frequencies moving towards the middle of larger substrates. This endoxylanase has five major subsites, with the catalytic site located between the third and fourth subsites, counting from the non-reducing end of the bound substrate. The subsite to the non-reducing ...


Mutagenesis Of The Active Site Of Glucoamylase From Aspergillus Awamori , Michael R. Sierks Jan 1988

Mutagenesis Of The Active Site Of Glucoamylase From Aspergillus Awamori , Michael R. Sierks

Retrospective Theses and Dissertations

The Aspergillus awamori glucoamylase gene was modified by cassette mutagenesis, and eleven mutated enzymes were expressed by Saccharomyces cerevisiae;Four mutations were constructed to change the essential Trp 120 residue in subsite 4 to Tyr, Phe, His, and Leu. All the mutations bound maltose (G2) and isomaltose (iG2) more strongly than wild-type glucoamylase. A subsite map of the Tyr 120 mutation showed significantly higher affinities for D-glucosyl residues in subsites 1 and 2, suggesting an interaction of Trp 120 with residues located there;Three carboxylic acid residues in the active site, Asp 176, Glu 179, and Glu 180, were mutated ...


A Nonequilibrium Thermodynamic Approach To Biological Energy Conversion Systems , Michael K. Dowd Jan 1986

A Nonequilibrium Thermodynamic Approach To Biological Energy Conversion Systems , Michael K. Dowd

Retrospective Theses and Dissertations

Energy conversion devices are commonly built from individual subunits in order to increase the force or flow that can be obtained from the device. Examples occur in both engineering and biology and include the cylinders of an internal combustion engine, the plates of a battery, the cross-bridges of muscle, and the active transport complexes in a cell membrane;This work describes the behavior of assemblies of individual energy converting subunits. The linear phenomenological laws of nonequilibrium thermodynamics are used as constitutional equations that describe the relationship between the forces and flows of a subunit. These relationships along with the restrictions ...