The Escherichia coli (E. coli) outer membrane protease OmpT is an endopeptidase of the omptin family in gram negative bacteria. OmpT cleave preferentially between two consecutive basic residues, especially Arg-Arg, and it has been classified as an aspartyl protease based on its crystal structure although biochemical confirmation of a catalytic aspartyl residue is lacking (Vandeputte-Rutten, et al., 2001). Our lab has successfully engineered the P1 and P1’ specificity and selectivity of OmpT by employing novel strategies for the isolation of enzyme variants that cleave desired substrates from large combinatorial libraries screened by flow cytometry. However, the engineering of proteases with altered specificity beyond the P1 and P1’ residues of the substrate have not been demonstrated. By applying high throughput screening of large libraries of OmpT constructed by structure-guided saturation mutagenesis of the S2 subsite (which recognizes the P2 residue), as well as random mutagenesis by error prone viii PCR and DNA shuffling, we engineered an OmpT variant exhibiting about 56 fold change in the selectivity for the P2 position in peptide substrates. Specifically, this enzyme preferred an acidic residue (Glu) over Tyr which is preferred by the wild type OmpT. Molecular modeling was then employed to provide insights on how mutations in OmpT mediated this change in P2 specificity. A long term goal of protease engineering is to generate highly specific enzyme variants that can be used for the irreversible inactivation of disease targets. The anaphylatoxin C3a is a key mediator in inflammation and has been implicated with multiple inflammatory diseases. Since the site of anaphylatoxin C3a recognized by cellular receptors lie in its C-terminus, a protease cleaving the C-terminus of C3a could be therapeutically relevant. Using high throughput screening and directed evolution we successful isolated C3a cleaving enzyme variants and have characterized them biochemically. Finally as part of this dissertation we have employed high throughput screening methods to dissect the substrate specificity of members of the kallikrein family of mammalian proteases which are implicated in a number of physiological and disease functions. The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction and vascular cell growth. KLK6 is over-expressed in breast and ovarian cancer tissues and has been shown to cleave peptides derived from human ix myelin protein and the Aβamyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6 by substrate phage-display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin-and chymotrypsin-like selectivities with Tyr/Arg preferred at the P1 site, Ser/Arg strongly preferred at P1’ and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1’. Docking simulations of consensus peptide substrates was used to infer possible identities of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates such as ionotropic glutamate receptor (GluR) and synphilin. / text

Aim of this study was to select environmental E. coli isolates that produced colicins against the swine pathogen E. coli K88+. In initial evaluation using a modified plate method with 18 colicinogenic E. coli constructs, colicins E3, E4, E5, E9, Ia, K and N were found to possess inhibitory activity against 12 ETEC K88+ strains. A total of 463 environmental isolates from cattle rumen, cattle feces, pig feces and hog manure-amended soil were screened for colicin production by a modified plate test. Further, colicinogenic isolates were screened for five toxin genes LT, STa, STb, VT1 and VT2 as well as K88 (F4) fimbriae using PCR reactions. Fourteen non-pathogenic isolates were subjected to characterization of colicin genes by PCR using 9 new primer sequences, antibiotic susceptibilities and substrate utilization. Two potential probiotic strains of E. coli, UM-2 and UM-7 which produced colicins that could utilize potato starch and inulin were selected for in-vitro competition with E. coli K88+ strain 2-12. In vitro competition between the synbiotics and E. coli K88+ revealed inhibition of E. coli K88+. Based on the present in vitro studies it could be concluded that carefully selected potential synbiotics should be further studied for their role in protecting piglets from post-weaning diarrhea without antibiotics.

Aim of this study was to select environmental E. coli isolates that produced colicins against the swine pathogen E. coli K88+. In initial evaluation using a modified plate method with 18 colicinogenic E. coli constructs, colicins E3, E4, E5, E9, Ia, K and N were found to possess inhibitory activity against 12 ETEC K88+ strains. A total of 463 environmental isolates from cattle rumen, cattle feces, pig feces and hog manure-amended soil were screened for colicin production by a modified plate test. Further, colicinogenic isolates were screened for five toxin genes LT, STa, STb, VT1 and VT2 as well as K88 (F4) fimbriae using PCR reactions. Fourteen non-pathogenic isolates were subjected to characterization of colicin genes by PCR using 9 new primer sequences, antibiotic susceptibilities and substrate utilization. Two potential probiotic strains of E. coli, UM-2 and UM-7 which produced colicins that could utilize potato starch and inulin were selected for in-vitro competition with E. coli K88+ strain 2-12. In vitro competition between the synbiotics and E. coli K88+ revealed inhibition of E. coli K88+. Based on the present in vitro studies it could be concluded that carefully selected potential synbiotics should be further studied for their role in protecting piglets from post-weaning diarrhea without antibiotics.

Thesis (doctoral)--Rijksuniversiteit te Groningen, 1983. / Summary and foreword in Dutch. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

A Prolactina (PRL) é um dos hormônios mais versáteis em termos de ação biológica. Sua ação mais conhecida está relacionada com o estímulo da lactação e regulação do crescimento e da diferenciação da glândula mamária; também apresenta importante aplicação diagnóstica. Somando os crescentes estudos sobre suas possíveis aplicações terapêuticas, fica cada vez mais notória a necessidade da obtenção desse hormônio puro, biologicamente ativo e na sua forma autêntica.O objetivo fundamental desse projeto foi a produção de hPRL em escala laboratorial a partir de bactérias (E.coli) modificadas geneticamente, utilizando um sistema de expressão baseado no promotor Lambda () PL, o mesmo utilizado com sucesso em nosso laboratório na expressão do hGH. Descrevemos nesse trabalho um processo de cultivo em biorreator, onde não foi utilizado o repressor cIts, uma proteína termo-sensível que usualmente é utilizada para inibir o funcionamento do promotor PL durante crescimento a 30ºC. O processo de cultivo apresenta basicamente três etapas: na primeira etapa o crescimento é realizado sem adição contínua de nutrientes (cultivo em batch), na segunda etapa ocorre adição contínua de nutrientes e carboidrato (cultivo em fed-batch) e na última etapa é realizada a ativação, caracterizada pelo aumento da temperatura mantendo-se a adição de nutrientes e carboidrato. Esse processo de fermentação rápido e flexível, com duração média de 20 horas, permitiu obter uma biomassa final correspondente à densidade óptica de aproximadamente 30 A600nm (unidades ópticas de absorbância em 600nm) e com uma expressão da ordem de 1g de hPRL mL-1 A600 -1, as mais altas já relatadas para secreção de prolactina no espaço periplásmico. A hPRL monomérica foi purificada e caracterizada por métodos físico-químicos e biológicos, os quais confirmaram a sua atividade biológica e imunológica, o seu correto processamento e uma massa molecular relativa (Mr) de 22.906. / Prolactin (PRL) is one of the most versatile hormones in terms of biological action. His best known action is related to the stimulation of lactation and regulation of growth and differentiation of the mammary gland; it also has wide important diagnostic applications. Considering all the increasing studies on its potential therapeutic applications, the need for obtaining this hormone in its pure, biologically active and authentic form becomes clearer and clearer. The fundamental objective of this project was the production of hPRL on the laboratory scale, from genetically modified bacteria (E.coli), using an expression system based on Lambda () PL promoter, the same successfully used in our laboratory for the expression of hGH. We set up a cultivation process in bioreactor, where the repressor (cIts), a thermo-sensitive protein that is usually used to inhibit the PL promoter during the growth phase (30°C). The cultivation process presents basically three stages: the first step in was not used the growth is carried out without the continuous addition of nutrients (batch cultivation), the second step in which a continuous addition of nutrients and carbohydrate occurs (fed-batch cultivation) and a final step when activation is carried out. The latter is characterized by an increased temperature, still maintaining the addition of nutrients and carbohydrate. This fast and flexible process of fermentation, with the average duration of 20 hours, led to a final biomass of approximately 30 A600nm (units of optical absorbance at 600nm), with the expression of about 1g of hPRL mL-1A600 -1, the highest ever reported for the secretion of prolactin in the periplasmic space. Monomeric hPRL was purified and characterized by physical-chemical methods and biological assays, which confirmed its biological and immunological activity, correct processing and a relative molecular mass (Mr) of 22,906.

No description available.

Genomic data has led to the realization that nearly one third of the bacterial genes discovered have unknown functions. Promising drug targets are no doubt among the conserved and essential genes, but drug development awaits an understanding of the basic physiological roles of the cryptic bacterial proteins they encode. The pressing need for novel antibiotics mandates investigation of these essential bacterial genes of unknown function. For a subset of such genes, it is becoming increasingly apparent that they may play critical roles in ribosome assembly or function in bacteria. One such essential gene, encoding the putative GTPase YihA in Escherichia coli has been highlighted as a target of particular importance for study (Galperin M.Y. and E.V. Koonin. (2004). 'Conserved hypothetical' proteins: prioritization of targets for experimental study. Nucleic Acids Res 32(18): 5452-63.). To study yihA's function, a strain with yihA under P_BAD control was generated and used to examine the impact of the loss of the gene product on ribosome assembly. Depletion of the YihA protein was found to cause accumulation of 30S and 50S subunits at the expense of 70S ribosomes, and the formation of long filamentous cells apparently incapable of dividing. Chemical genetic interactions were identified through chemical synthetic lethality screening with antibiotics which target the ribosomal A -site, and antibiotics which affect the bacterial cell wall. Finally, investigation of the importance of several patches of basic residues of YihA through mutagenesis and in vivo complementation shows the protein to be tolerant of mutation to non-catalytic residues. The inability of mutants with changes to amino acids important for nucleotide binding to complement represents the strongest evidence to date that the YihA protein relies on GTP binding and hydrolysis as part of its essential function in vivo. The findings of this study support a role for YihA in the poorly understood process of ribosome assembly in vivo, and represent important progress towards realization of this essential process as a source of novel antibiotic targets. / Thesis / Master of Science (MS)

No description available.

No description available.