Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Alan M. Lambowitz. Vita. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Thesis (D.Biotech.) - University of Queensland, 2003. / Includes bibliography.

Thesis (M.A.)--Trinity Graduate School, 2007. / Abstract. Includes bibliographical references (leaves 151-159).

Thesis (M.S.)--Rowan University, 2009. / Typescript. Includes bibliographical references.

Thesis (MSc)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (Ph.D.)--University of North Carolina at Greensboro, 2007. / Title from PDF t.p. (viewed Oct. 22, 2007). Directed by Nancy Myers; submitted to the Dept. of English. Includes bibliographical references (p. 209-228).

Thesis (M.A.)--Trinity Graduate School, 2007. / Abstract. Includes bibliographical references (leaves 151-159).

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins and has been involved in processes such as DNA repair and gene expression. The poly(ADP-ribose) polymer (PAR) is mainly catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). TcPARP of Trypanosoma cruzi, appears to play a role in DNA repair mechanism during Chagas’ disease and believed to be involved in controlling different phases of cell growth. In this study, cloning and characterization of mutants of TcPARP is reported. In first phase, five mutants of TcPARP, already cloned in pET-22b+, were expressed BL21 Rosetta2(DE3). Since expression in this vector was poor, all mutants were cloned in pNH-TrxT vector and expressed in BL21 Rosetta2(DE3). Protein purification of mutant TCP-c006 (124 residues deleted from N-terminus) was performed using His6 tagged FF crude IMAC column followed by gel filtration. Fluorescence-based activity assay of mutant TCP-c006 shows only 35% conversion of NAD+ to nictonamide compared to wild type. Moreover 50 times more protein is required for this conversion when compared with wild type. During DNA binding assay (EMSA), mutant TCP-c006 did not bind with DNA, clearly indicating that N-terminus is necessary for DNA binding and activity of TcPARP. In future, solubility of both wild type and mutants of TcPARP in different expression host system using various fusions such as MBP, T4 lysozyme etc could be tested. Structure of both wild type and mutants of TcPARP could be determined by using Static light scattering (SLS).

The purity of heterologous recombinant proteins is of utmost importance to the pharmaceutical sector since most are consumed as therapeutic agents by humans. Variability caused by co- and posttranslational modifications is a major concern in pharmaceutical production. In order to develop strategies which guarantee a homogeneous product in a robust production process, it is important to better understand the metabolic basis of the synthesis of related non-canonical amino acids. So far, studies have identified high glucose fluxes in connection to oxygen limitation and overexpression of leucine-rich proteins as possible reasons for the production of non-canonical amino acids and their incorporation into heterologous proteins expressed in Escherichia coli. The results presented in this work provide evidence that oscillations in the concentrations of glucose and oxygen as they occur in inhomogeneous industrial scale bioreactors potentiate the synthesis and incorporation of norvaline into the leucine-rich protein IL-2, heterologously expressed in E. coli W3110M, as observed in well-mixed homogenous cultures and perturbed shake flask cultivations. In order to represent the heterogeneities existing in large-scale bioreactors, two experimental setups were applied, using a simple shake flask scale-down model developed to monitor dissolved oxygen and pH online during a batch and fed-batch cultivation phases. Results here show that by applying repeated glucose pulses to the glucose limited culture, which consequently induce oscillations in dissolved oxygen, norvaline is accumulated. Analysis of inclusion bodies that resulted from the expressed IL-2 revealed the presence of norvaline in the protein. A higher concentration of norvaline was observed in the oscillating scale-down model compared to the non-perturbed culture, which suggests that the conditions as they typically occur in large scale bioreactors may be critical for product quality. The results and tools, developed in this work are a solid basis for future cell engineering approaches to overcome the challenges in view of product quality.