Browsing by Author "Sines, Brian James"

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    Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents
    Sines, Brian James (Virginia Tech, 2000-11-03)
    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.
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    Isolation and partial characterization of a water stress protein of the desiccation-tolerant cyanobacterium Nostoc commune UTEX 584 expressed in Escherichia coli
    Sines, Brian James (Virginia Tech, 1996)
    A desiccation-tolerant cyanobacterium Nostoc commune accumulates a novel group of water stress proteins (Wsp) in response to cycles of repeated drying and rehydration. Antibodies, specific for Wsp, were used to screen a lambdafix II library of N. commune UTEX 584 Bam H1 DNA fragments and an 8.5-kb fragment, containing a gene cluster that synthesized a 59-kDa cross-reactive protein. The cloned fragment comprised five ORF’s. The ORF’s 59, 24, 22, 36, and 70, each potentially encode products of molecular weights of 59, 24, 22, 36, and 70-kDa, respectively. The 59 and 24 ORF products were found to be expressed in E. coli. The 59-kDa product of this fragment gives the strongest cross-reaction with the Wsp antiserum. The 59-kDa protein was partially purified. The 24-kDa product was successfully purified to homogeneity and partially characterized. This study used E. coli strain DH10B transformed with the pTrc 99A plasmid. The pTre 99A contains the 8.5-kb gene cluster fragment of interest. The products of ORF 24 and 59 were isolated using an initial 40-60 % ammonium sulfate precipitation of a clarified E. coli cell lysate. The clarified cell lysate was then subjected to streptomycin sulfate precipitation. The cell lysate was then dialyzed extensively. The cell lysate was then applied to a Mono Q HR 5/5 anion exchange column using a 2 M KCl gradient elution procedure. The Mono Q column yielded a fraction containing both ORF products which eluted with approximately 400 mM KCl. This fraction was then applied to a Superose 12 HR 10/30 gel filtration column. The eluent fraction containing the ORF 24 product was then reapplied to the Superose 12 to yield the final fraction containing only the ORF 24 product. The final fraction of ORF 24 was purified to homogeneity as determined by SDS-PAGE analysis. Approximately 750 μg of ORF 24 was isolated. This preparation was used for characterization studies. Characterization studies of ORF 24 consisted of an amino-terminal sequence analysis, an estimation of the molecular weight using gel filtration chromatography and SDS-PAGE analysis, and an analysis of enzymatic activity as suggested by amino acid sequence homologies. The amino-terminal sequence of ORF 24 is P V E Q R S H D. The molecular weight of ORF 24 using gel-filtration chromatography and SDS-PAGE analysis is 26-kDa and 23-kDa, respectively. From gene sequence analysis, the molecular weight of ORF 24 is known to be 24,340-Da. These data indicate that ORF 24 is a monomer. ORF 24 was found to have amino acid sequence homologies with a pectate lyase (E 4.2.2.2) periplasmic precursor from Erwinia caratovora subspecies and a dextransucrase (EC 2.4.1.5) precursor from Streptoccocus mutans GS-5. However, pectate lyase activity was not detected in cellular extracts over a 24 hour period. In addition, ORF 24 was not found to interact with 10 % substrate solutions of N-acetylglucosamine, pectin, UTEX 584 sheath material, DRH1 sheath material, sucrose, or glucose using thin layer chromatography. These studies indicate that the enzymatic activities proposed from amino acid sequence homologies have not been detected. The suggestion that ORF 24 is a water stress protein with a protective function on a structural level with regards to desiccation-tolerance requires further study.