Thesis (Ph.D.)--University of Nebraska-Lincoln, 2009. / Title from title screen (site viewed July 21, 2009). PDF text:xiii, 231 p. : ill. ; 4 Mb. UMI publication number: AAT 3350376. Includes bibliographical references. Also available in microfilm and microfiche formats.

Graduation date: 2005

Caryolanemagnolol and clovanemagnolol promote neuronal regeneration in various cell and animal based assays. The protein targets of these natural products are not currently known. Derivatives of caryolanemagnolol and clovanemagnolol were synthesized for the purpose of affinity chromatography. The derivatives are accessed rapidly through optimized procedures. / text

Thesis (M.S.)--Ohio University, August, 2000. / Title from PDF t.p.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 106-107). Also available via the Internet.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed Aug. 1, 2007). PDF text: 1 v. : ill. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed Feb. 17, 2009). PDF text: ix, 137 p. : ill. (some col.) ; 2 Mb. UMI publication number: AAT 3325856. Includes bibliographical references. Also available in microfilm and microfiche formats.

Insufficient supply and pathogen safety concerns regarding plasma-derived therapeutic proteins, such as fibrinogen and immunoglobulins, have been the impetus for the development of genetic engineering techniques and separations methods for the economical and safe production of these proteins. This study is concerned with the isolation of these important therapeutics from complex media. Immunoaffinity chromatography has been an important method in the isolation of these products, typically being implemented as the final cleanup step yielding an extremely pure, homogenous final product. However, the use of immunoaffinity chromatography in large-scale purification processes have been precluded due to high capital costs and the inherent lability of immunosorbents. Peptide-based affinity sorbents are being developed in order to surmount the inherent limitations posed by monoclonal antibodies that are used as ligands in immunosorbents. The objective of this research is to quantitatively assess the impact of affinity ligand orientation, local density and transport phenomena on peptide-based affnity sorbent performance. The peptides under study herein can form high-affinity complexes with their protein targets, thus these ligands are one of the newest technologies arising from combinatorial chemistry with applications to the difficult problem of purifying high-molecular weight proteins from complex mixtures. Two types of structural motifs which are common to small peptide affinity ligands derived from combinatorial chemistry are studied here: a linear peptide which is comprised of the affinity recognition sequence in its entirety and a chain structure which displays multiple branches of the recognition sequence emanating from a central lysinic core structure. Two recognition sequences are studied here which bind plasma proteins. One peptide recognition sequence forms a high affinity complex with fibrinogen. Another peptide recognition sequence binds the Fc region of immunoglobulins. Immunglobulins are plasma proteins which range in molecular weight from 155 to 900-kDa and are valuable for therapeutic uses for imparting passive immunity. This study seeks to identify factors analogous to those manifested in immunosorbent performance that may also be important in the optimal design of peptide-based affinity sorbents. In general, previous research with the design of immunosorbents have found that immunosorbent performance, i.e., target-binding efficiency or activity, is substantially dependent upon several factors which include effects associated with ligand orientation, and local density as related to steric incumbrance of target binding sites, and transport phenomena as related to under utilization of intra matrix volume. In summary, this study asks the questions: (1) What factors regarding ligand orientation, local ligand density, and intraparticle transport phenomena, are important in the optimal design of peptide affinity sorbents?; and (2) Are these effects analogous to those manifested in immunosorbent performance? This study seeks to investigate the use of techniques used to mitigate the effects associated with these negative factors upon immunosorbent performance in order to elucidate the nature of these same effects upon peptide-based affinity sorbents. For example, oriented ligand immobilization can be facilitated through selective coupling chemistries and the premasking of ligand binding domains prior to immobilization. In addition, the manipulation of local ligand density using novel spatially controlled matrix activation and ligand immobilization methods can be assessed and implemented for the optimization of the performance and design of peptide-based affinity sorbents. This study has found that enhanced transport phenomena into the matrix interior volume can be achieved by using low solids content cellulose matrices having a low extent of crosslinking. This study demonstrates the effective use of these large-particle diameter, low-solids content cellulose hydrogel matrices in immunoaffinity, peptide-based affinity and ion exchange chromatography in the separation of high-molecular weight therapeutic proteins. / Ph. D. / This report presents an evaluation of the design of low-solids content, large-particle diameter beaded cellulose supports for column-mode protein purification. The study presented here optimizes the molecular accessibility of the cellulose support to high molecular weight proteins relative to the mechanical stability of the support at high operating linear velocities by the manipulation of bead particle diameter and solids content. A novel epoxidegradient activation method (epoxy-GAM) is developed for creating a gradient of support activation for support crosslinking and affinity ligand installation in the preparation of DEAE hydrogel matrices. These cellulose hydrogel supports were evaluated with regards to structure, dynamic and static binding capacity, pressure-flow stability, chemical stability and intraparticle transport phenomena. The utility of the low-solids content, large-particle diameter DEAE hydrogel matrices was demonstrated in a column-mode protein purification using albumin and fibrinogen mixtures.

No description available.

Antibodies represent the largest group of biopharmaceuticals. Due to the nature of their clinical applications, they often need to be produced in large quantities. Plants have distinct advantages of producing large quantities of recombinant proteins, and tobacco is arguably the most promising plant for plant-made-pharmaceuticals (PMP) due to its high biomass yields and robust transformation technology. However, to produce proteins using transgenic tobacco for human applications, purification of the proteins is challenging. On the other hand, Protein A, a bacterial cell wall protein isolated from Staphylococcus aureus that binds to the Fc regions of immunoglobulins, is useful to the isolation and purification of antibodies. An affinity chromatography purification step utilizing Protein A resin introduced early in the purification process can reduce successive unit operations, thereby reducing the overall process cost. However, directly applying tobacco extract to Protein A chromatography columns may be problematic due to the non-specific binding of native tobacco proteins (NTP). In this project, three different Protein A resins, ProSepvA High Capacity, ProSep-vA Ultra, and ProSep Ultra Plus, marketed by Millipore, were studied to provide valuable information for future downstream processes for antibody purification from transgenic tobacco. The efficiency of the post load wash buffer to reduce non-specific binding of NTP to the ProSep A resins were evaluated by altering the ionic strength and pH. Lower salt concentrations of sodium chloride (NaCl) in the post load wash preformed best at reducing the non-specific binding of NTP to the ProSep A resins, while higher salt concentrations were more effective at reducing the amount of NTP contaminants present during elution of the columns. Using a post load wash buffer with an intermediate pH between the binding buffer and the elution buffer was more efficient at eluting our model antibody, human IgG. However, lowering the ionic strength and the pH of the post load wash buffer resulted in a greater presence of IgG prematurely eluting from the ProSep A resins. The non-specific binding of NTP to the resins reduced the dynamic binding capacity (DBC) of the resins after repeated cycles of tobacco extract samples were loaded onto the column. Nevertheless, cleaning the columns with denaturing solutions, such as urea or guanidine hydrochloride, every 8-10 cycles was effective in regenerating the DBC of the resins and prolonging the life cycle of the resins. This is important to evaluating the economic feasibility of directly using Protein A chromatography to recover antibodies from tobacco extract. Of the three Protein A resins studied, ProSep Ultra Plus performed best for antibody purification from tobacco using a PBS wash buffer with a lower ionic strength of 140mM NaCl and an intermediate pH of 5. / Master of Science