Split-protein systems are invaluable tools used for the discovery and investigations of the complexities of protein functions and interactions. Split-protein systems rely on the non-covalent interactions of two fragments of a split protein to restore protein function. Because of this, they have the ability to restore protein functions post-translationally, thus allowing for quick and efficient responses to a milieu of cellular mechanisms. Despite this, split-protein systems have been largely limited as a reporting tool for protein-protein interactions. The recent discovery of inteins has the potential of broadening the scope of split-protein systems. Inteins are protein elements that possess the unique ability of post-translationally ligating protein fragments together with a native peptide bond, a process termed protein splicing. This allows split-proteins to reassemble in a more natural state. Exploiting this property and utilizing protein engineering techniques and methodologies, several approaches are described here for restoring and controlling split-protein functions using inteins. First, the protein splicing behaviour was demonstrated with the development of a simple in vitro visual fluorescence assay that relies on examining the subcellular localization of different fluorescent proteins. Inteins were then used to reassemble and restore function to artificially split genetically encoded Ca2+ indicators. Second, inteins were shown to be able to simultaneously restore protein function to two target proteins. The first target protein was restored through the normal protein splicing pathway while the second was restored through non-covalent interactions of the split-protein fragments. This is a previous unknown property of inteins. Lastly, an intein was engineered to respond to an external light-stimulus that triggered protein splicing to restore split-protein function. The photoactivatable intein, coupled with the versatility of light, allows exquisite control in both space and time for the restoration of protein function within cells. The modularity of the photoactivatable intein can be simply attached to a variety of split-proteins. This was demonstrated with the restoration of various split-protein functions.

Split-protein systems are invaluable tools used for the discovery and investigations of the complexities of protein functions and interactions. Split-protein systems rely on the non-covalent interactions of two fragments of a split protein to restore protein function. Because of this, they have the ability to restore protein functions post-translationally, thus allowing for quick and efficient responses to a milieu of cellular mechanisms. Despite this, split-protein systems have been largely limited as a reporting tool for protein-protein interactions. The recent discovery of inteins has the potential of broadening the scope of split-protein systems. Inteins are protein elements that possess the unique ability of post-translationally ligating protein fragments together with a native peptide bond, a process termed protein splicing. This allows split-proteins to reassemble in a more natural state. Exploiting this property and utilizing protein engineering techniques and methodologies, several approaches are described here for restoring and controlling split-protein functions using inteins. First, the protein splicing behaviour was demonstrated with the development of a simple in vitro visual fluorescence assay that relies on examining the subcellular localization of different fluorescent proteins. Inteins were then used to reassemble and restore function to artificially split genetically encoded Ca2+ indicators. Second, inteins were shown to be able to simultaneously restore protein function to two target proteins. The first target protein was restored through the normal protein splicing pathway while the second was restored through non-covalent interactions of the split-protein fragments. This is a previous unknown property of inteins. Lastly, an intein was engineered to respond to an external light-stimulus that triggered protein splicing to restore split-protein function. The photoactivatable intein, coupled with the versatility of light, allows exquisite control in both space and time for the restoration of protein function within cells. The modularity of the photoactivatable intein can be simply attached to a variety of split-proteins. This was demonstrated with the restoration of various split-protein functions.

The goal of this research is to understand how three different parameters affect single chain variable fragment (scFv) binding capacity. The parameters that were varied include the number of variable complementarity determining regions (CDRs), the number amino acids used to diversify CDRs, and configuration of the structure. How the parameters affect the binding capacity will be tested using the yeast two hybrid assay against five different protein domains. Eight scFv libraries were generated; the genes expressing the scFvs were constructed and the CDRs were randomized using PCR amplification. Genes expressing scFvs were cloned, using the homologous gap repair mechanism in <i>Saccharomyces cerevisiae</I>. Representative members of scFv libraries were sequenced to confirm correct construction.<p> Library diversity was calculated from the library transformation efficiency. Transformation efficiency refers to the number of cells that grew at the time of transformation of the scFv gene into yeast cells. There were significant differences in the diversity of the scFv libraries, which created difficulty in comparing the library binding capacities. Sequencing the scFv libraries revealed that on average 50% of each library contained correct scFv sequences. The percent of correct sequences within each library was then used to calculate the functional diversity.<p> The yeast two-hybrid assay was used to screen the scFv libraries for interactions and to test binding capacity. The binding capacity of the scFv libraries was tested and compared in five different yeast two-hybrid assays using five protein domains as the targets for each screen. The screening results showed that in all cases cyclic scFv libraries had a statistically significant higher binding capacity than linear scFv libraries despite a diversity bias against the cyclic libraries. There was no clear trend in binding capacity with the other two parameters; however, the four amino acid three CDR libraries dominated over the other libraries in almost every screen.<p> Some of the scFvs isolated from the screens were expressed in <i>E. coli</i> and <i>S. cerevisiae</i>to analyze for proper expression and correct size. All the scFvs that were isolated and analyzed were the correct size and could be purified using a poly histidine tag.<p> Due to its bioaffinity and specificity, scFvs were constructed to profile disease patterns, and to identify potential drug targets. In addition to its original application to health-related studies, scFvs could also be extended to locate potential metabolic bottlenecks, to alter metabolic flux to enhance productivity, and regulate metabolic bionetworks. Industrial microorganisms are generally carrying more than two sets of chromosomes, making it difficult to be genetically engineered when conventional approaches are employed. With the availability of scFvs as reported in this thesis, we are able to design specific scFvs that selectively bind to target proteins, resulting in re-routing of metabolic flux within the microorganism, toward a high productivity of desired product. ScFvs can be applied to industrial microorganisms directly, leading to the development of new fermentation processes.

The goal of this research is to understand how three different parameters affect single chain variable fragment (scFv) binding capacity. The parameters that were varied include the number of variable complementarity determining regions (CDRs), the number amino acids used to diversify CDRs, and configuration of the structure. How the parameters affect the binding capacity will be tested using the yeast two hybrid assay against five different protein domains. Eight scFv libraries were generated; the genes expressing the scFvs were constructed and the CDRs were randomized using PCR amplification. Genes expressing scFvs were cloned, using the homologous gap repair mechanism in <i>Saccharomyces cerevisiae</I>. Representative members of scFv libraries were sequenced to confirm correct construction.<p> Library diversity was calculated from the library transformation efficiency. Transformation efficiency refers to the number of cells that grew at the time of transformation of the scFv gene into yeast cells. There were significant differences in the diversity of the scFv libraries, which created difficulty in comparing the library binding capacities. Sequencing the scFv libraries revealed that on average 50% of each library contained correct scFv sequences. The percent of correct sequences within each library was then used to calculate the functional diversity.<p> The yeast two-hybrid assay was used to screen the scFv libraries for interactions and to test binding capacity. The binding capacity of the scFv libraries was tested and compared in five different yeast two-hybrid assays using five protein domains as the targets for each screen. The screening results showed that in all cases cyclic scFv libraries had a statistically significant higher binding capacity than linear scFv libraries despite a diversity bias against the cyclic libraries. There was no clear trend in binding capacity with the other two parameters; however, the four amino acid three CDR libraries dominated over the other libraries in almost every screen.<p> Some of the scFvs isolated from the screens were expressed in <i>E. coli</i> and <i>S. cerevisiae</i>to analyze for proper expression and correct size. All the scFvs that were isolated and analyzed were the correct size and could be purified using a poly histidine tag.<p> Due to its bioaffinity and specificity, scFvs were constructed to profile disease patterns, and to identify potential drug targets. In addition to its original application to health-related studies, scFvs could also be extended to locate potential metabolic bottlenecks, to alter metabolic flux to enhance productivity, and regulate metabolic bionetworks. Industrial microorganisms are generally carrying more than two sets of chromosomes, making it difficult to be genetically engineered when conventional approaches are employed. With the availability of scFvs as reported in this thesis, we are able to design specific scFvs that selectively bind to target proteins, resulting in re-routing of metabolic flux within the microorganism, toward a high productivity of desired product. ScFvs can be applied to industrial microorganisms directly, leading to the development of new fermentation processes.

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<p> Proteins vary tremendously in many of their physical and chemical properties. In order to perform in vitro application or analysis, one protein must be separated from other cellular components. This process is called protein purification. With the advances of modem science and technology, many protein purification schemes have been developed. Among them, the ELP-intein protein purification system has recently attracted an increasing amount of attention because of its positive characteristics: it is simple, inexpensive, scalable, with a high throughput, protease-free, etc. However, although the scientific literature reports all those good aspects of the system, several bad responses to it still exist. In this thesis, through comparing expression and purification of four different self-cleavage ELP fusion proteins, we propose a general solution to these problems for the first time. This makes a significant contribution to increased utility of the method of protein purification using self-cleavable stimulus responsive tags. </p> <p> When ELP-intein fusion proteins are expressed in bacteria, formation of non-native cytoplasmic aggregates (inclusion bodies) is a common problem which affects the yield of target protein. Inclusion bodies are generally assumed to contain misfolded or partially folded protein through exposure of hydrophobic patches and the consequent intermolecular interactions. Despite a loss of total expression yield and the need for more time, culturing at a lower temperature was reported to promote the expression of genes into soluble proteins and alleviate IB formation. Directly motivated by previous reports, we have applied a low-temperature expression strategy to solve the problems in this research. As expected, most of the T4-ELP inclusion bodies disappeared, and were transformed into a soluble expression, when culturing at lower temperatures. </p> <p> Inverse transition cycling (lTC), as the core method for the system we investigated has proved successful in the past with proteins that were expressed to high levels. However poor level ELP-intein tagged protein expression happens from time to time. It is hypothesized that if an ELP tagged molecule is present in a solution at a very low concentration, adding an excess amount of free ELP to the sample would form hybrid aggregates via the interaction of ELP moieties of the two molecules. We used this efficient and reversible capture system for low yield recombinant protein purification, and found it is perform very well. </p> / Thesis / Master of Applied Science (MASc)

Orientador: Eduardo Bagagli / Resumo: Os dermatófitos são um grupo de fungos constituídos pelos gêneros Trichophyton, Epidermophyton e Microsporum que têm a habilidade de degradar a queratina. É por essa razão que podem colonizar a pele do homem e dos animais, embora também possam crescer no ambiente, geralmente em solos com restos de queratina. As características morfológicas permitem a identificação e classificação taxonômica das espécies, mas realizar um diagnóstico baseado nestas características pode levar a erros. As técnicas moleculares ajudam a realizar diagnósticos mais rápidos baseados em uma identificação molecular e também têm possibilitado importantes avanços quanto aos estudos filogenéticos, sendo as sequências ITS1-5.8S-ITS2 as mais utilizadas. No entanto, estes marcadores não são suficientes para identificar e elucidar todas as relações filogenéticas e taxonômicas dos dermatófitos devido à ampla variedade de espécies existentes, -sendo necessário novos marcadores genéticos, como o intein PRP8. Os inteins são elementos genéticos parasitas, de natureza proteica, presentes em alguns fungos e estão associados a importantes genes altamente conservados. Ointein PRP8 está localizado nogenePRP8 que codifica para a proteína PRP8 associada ao complexo do spliciosoma. O presente estudo visa caracterizar o intein PRP8 em fungos dermatófitos, de forma a empregar estes elementos genéticos como marcadores moleculares para uma correta identificação destas espécies e elucidações das relações filogenéticas do grupo.... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Dermatophytes are a fungal group composed by the genera Trichophyton, Epidermophyton and Microsporum with the ability to degrade keratin. That is why they can colonize the skin of man and animals, although they can also grow in the environment, usually in soils with remains of keratin. The morphological characteristics allow the identification and taxonomic classification of the species, but a diagnosis based on these characteristics is difficult. Molecular techniques made diagnoses based on molecular identification faster and made possible important advances in phylogenetic studies, with ITS1-5.8s-ITS2 sequences being the most used. However, these markers are not enough to elucidate all the phylogenetic relationships and taxonomic of dermatophytes due to the wide variety of existing species, and new genetic markers may be needed for correct identification and phylogenetic studies, such as the PRP8 intein. Inteins are parasitic genetic elements of a protein nature present in some fungi and are associated with important highly conserved genes. The PRP8 intein is located within the PRP8 gene coding for the PRP8 protein associated with the spliciosome complex. The present study aims to characterize the PRP8 intein in dermatophyte fungi, in order to use these genetic elements as molecular markers for a correct identification of these species and elucidations of the phylogenetic relationships of the group. To accomplish this goal, 45 strains were molecularly characterized using th... (Complete abstract click electronic access below) / Mestre

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Spider silk has long been a subject of scientific research due to its remarkable mechanical properties. Until recently, there has been no way to effectively obtain spider silk except by harvesting it from individual spiders. With advances in technology, the genes that code for the individual spider silk proteins have been isolated and genetically engineered into other hosts to produce recombinant spider silk proteins (rSSp) of varying sizes, Larger rSSp have correspondingly greater mechanical properties in any resulting materials. Using current production methods, larger rSSp cannot be produced in commercially viable quantities while simultaneously being economically viable. The current production methods have shown that small rSSp are easier to produce and purify in genetically engineered systems while maintaining favorable yields. After the small molecular weight rSSp were expressed and purified, they were polymerized to form larger molecular weight rSSp, while having maintained mechanical properties of similarly sized rSSp from other expression systems. To accomplish this polymerization, two systems were designed that can catalyze this reaction: using a Spy System and an intein system. These two systems require no external cofactors or enzymes and occur spontaneously once initiated. The expression and purification of rSSp from both of these systems has been characterized. The Spy System did not produce high enough quantities of rSSp to be economically viable. Whereas the intein system produced yields of 5 g/L, which is higher than previously reported. The rSSp from the intein system have been made into biomaterials, such as films, hydrogels, and aerogels. The mechanical properties of these biomaterials were comparable to biomaterials from other spider silk protein production. Utilizing the intein system, projected cost estimates for the production of rSSp has been lowered from $350 to $40 per kilogram. This decreased cost of rSSp would allow a wider array of commercial applications.