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.

Rotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular calcium through multiple mechanisms that promote abnormal ion transport and subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially interact with model membranes mimicking caveolae in lipid composition and radius of curvature. Our laboratory has recently reported the colocalization and coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae. Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least one terminus were capable of binding the NSP4 bait. An in vitro binding assay confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1 residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates NSP4 signaling and/or intracellular trafficking.

Universiẗat, Diss., 2005--Freiburg (Breisgau). / Erscheinungsjahr an der Haupttitelstelle: 2004.

Human papilloma virus (HPV) is a ubiquitous virus implicated in a growing list of cancers, particularly cervical cancer‐ the second most common cancer among women worldwide. Although persistent infection with high‐risk oncogenic HPVs such as types ‐16 or ‐18 is necessary, additional factors like co‐infection with other viruses can play a role in cancer progression. Protein‐protein interactions play a central role in the infection, survival and proliferation of the virus in the host. Although some interactions of HPV proteins are well characterised, it is essential to discover other key viral interactions to further improve our understanding of the virus and to use this knowledge for the development of newer biomarkers and therapeutics. The aim of this study was to systematically analyse the interactions of HPV‐16 proteins using yeast two‐hybrid (Y2H). To achieve this, a clone collection of the viral proteome was generated by recombinatorial cloning and three independent Y2H screens were performed: (i) Intra‐viral screen to identify interactions among the HPV‐16 proteins; (ii) Inter‐viral screen to identify interactions with proteins of Herpes Simplex Virus (HSV) which is suggested to be a co‐factor; and (iii) Virus‐host screen to identify novel cellular binding partners. The intra‐viral Y2H screen confirmed some of the previously known interactions and also identified binding of the E1 and E7 proteins. Deletion mutagenesis was performed to map the interaction domains to the amino‐terminal 92 amino acids of E1 and carboxy‐terminal CxxC domain of E7. Replication assays suggest a possible repression of E1‐mediated episomal replication by direct binding of E7. The inter‐viral Y2H screen identified interactions of HPV proteins with seventeen HSV‐1 proteins including transcriptional regulator ICP4 and neurovirulance factor ICP34.5. The biological relevance of these interactions in the context of co‐infection is discussed. The virus‐host screen performed against a human cDNA library identified 54 interactions, a subset of which was validated by biochemical pull‐down assays. The functional relevance of an interaction between E7 and a proto‐oncogene spermatogenic leucine zipper protein (SPZ1) was further investigated suggesting a role of SPZ1 in E7‐mediated cell proliferation. The work presented in this thesis identifies several novel interactions of HPV proteins. Future work will involve the in‐depth elucidation of biological relevance of these interactions. In particular, the interactions of E7 with E1 and SPZ1 are of great interest to improve our understanding of the life cycle and pathogenesis of the virus which can be applied for improved strategies of prevention and treatment of malignancies caused by HPV.

The availability of cost-effective high throughput screening assays combined with an enhanced understanding of oncogenesis has driven the development of more potent, specific, and less toxic anti-cancer agents. At the forefront of these advances are immunoglobulin molecules and their fragments. However, difficulties in producing antibodies in sufficient quantity and quality for commercial application have driven the development of alternative systems that can produce antibodies efficiently and cost-effectively. This thesis focuses on the engineering of an antibody fragment referred to as a single chain variable fragment (scFv), which consists of antibody light and heavy chain variable domains fused together by a peptide linker. Although the use of scFvs circumvents many of the issue of full-length antibody production, they still possess their own unique set of difficulties, including stability. In this thesis, we explored the following strategies to increase scFv stability. First, we increased the number of linkers used to join the variable light and heavy domains. We constructed two linear and two cyclic permutated scFvs that contained additional peptide linkers. Two linear permutated scFvs, named Model 1 and Model 3, showed increased stability with calculated melting temperatures (Tms) exceeding that of the unpermutated scFv. The two cyclic scFvs were less stable with Tms less than that of the unpermutated scFv. Second, we mutated light and heavy variable domains by introducing prolines or mutating glycine to alanine in the variable domain framework regions. Sites for proline mutations and glycine to alanine mutations were identified and scFvs containing the mutations were purified and their thermal stability tested. Unfortunately, there were no discernible differences between purified scFv mutants and the control scFv. Third, we designed a new selection/screening strategy using phage display and yeast two-hybrid assays to identify complementarity determining regions on scFvs that increased intracellular stability. We used this strategy to isolate anti-Abl-SH3 scFvs. Transient expression of scFvs in K562 cells indicated that two anti-Abl-SH3 scFv decreased viability.

The herpesvirus life cycle involves a step where newly formed capsids leave the nucleus by translocating across the intact nuclear envelope (NE). Little is known about the role of cellular factors during nuclear egress. We sought to identify novel cellular proteins that interact with the conserved herpes simplex virus-1 (HSV-1) pUL34 by performing a yeast two-hybrid screen. pUL34 was chosen due to its crucial and multifunctional role during nuclear egress. From 42 cellular factors that interacted with pUL34 in yeast, twelve were further evaluated in mammalian cells by co-localization studies using immunofluorescence. No specific co-location between the tested cellular factors and pUL34 was observed in infected cells, thus the screen failed to convincingly identify novel pUL34 interactors. In the second part of the thesis we addressed the functional significance of the cellular protein torsinA (TA) in the HSV-1 life cycle. We became interested in TA due to its role in maintaining normal NE morphology. We showed that perturbing the normal function of TA through overexpression impaired HSV-1 replication and caused a defect in capsid nuclear egress. In mouse embryonic fibroblasts that failed to express TA (TA-/-MEFs), HSV-1 replication was also inhibited, but a defect in capsid nuclear egress was not apparent. Strikingly, infection in TA-null MEFs induced a NE breakdown, the extent of which was dependent on viral products involved in nuclear egress. The viral growth defect and NE envelope breakdown, however, seem to be TA-null cell line specific rather than a functional consequence of TA loss as indicated by TA-/-MEFs reconstituted with TA and 293T with reduced TA levels. In conclusion, overexpression and loss of TA have different effects on the HSV-1 life cycle.

<p> Bacterial cell division is an essential process, which is initiated by forming the Z-ring as a cytoskeletal scaffold at the midcell site, followed by the recruitment of a series of divisome proteins. In <i>Escherichia coli</i> (Ec), at least 15 divisome proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsB, FtsL, FtsI, FtsW, FtsN, FtsE, FtsX, ZapA, AmiC, EnvC) have been implicated in this process. The components of the cell division machinery proteins in <i>Neisseria gonorrhoeae</i> (Ng) differs from <i>E. coli. N. gonorrhoeae</i> possesses FtsA, but lacks FtsB. ZipA and FtsL in <i>N. gonorrhoeae</i> have low identity to ZipA and FtsL from <i>E. coli</i>. Our laboratory has studied the central division protein FtsZ in <i>N. gonorrhoeae</i>. Thus, my research investigated the role of <i>N. gonorrhoeae</i> FtsA in cell division and investigated the interactions between divisome proteins from <i>N. gonorrhoeae</i> to understand divisome assembly.</p> <p>This study determined the association of FtsA_Ng with FtsZ</sub>Ng and other divisome proteins in <i>N. gonorrhoeae</i> and identified the functional domains of FtsA<sun>Ng</sub> involved in these interactions using a bacterial two-hybrid (B2H) assay. FtsA_Ng interacted with FtsZ_Ng, FtsK_Ng, FtsW_Ng, FtsQ_Ng, and FtsN_Ng. Self-interactions of FtsA_Ng and FtsZ_Ng were also detected. FtsI_Ng, FtsE_Ng and FtsX_Ng did not interact with FtsA_Ng. The 2A₁, 2A₂ and 2B domains of FtsA_Ng were sufficient to interact with FtsZ_Ng independently. Domain 2A₁ interacted with FtsK_Ng and FtsN_Ng. Domain 2B of FtsA_Ng interacted with FtsK_Ng, FtsQ_Ng, and FtsN_Ng. Domain 2A₂ of FtsA_Ng interacted with FtsQ_Ng, FtsW_Ng, and FtsN_Ng. These data suggest that FtsA in <i>N. gonorrhoeae</i> plays a key role in interactions with FtsZ and other divisome proteins.</p> <p>The potential interactions between divisome proteins in <i>N. gonorrhoeae</i> were examined using B2H assays. The comparisons between the <i>N. gonorrhoeae</i> divisome protein interaction network and those of <i>E. coli</i> and <i>S. pneumoniae</i> indicates that the divisome protein interactome of <i>N. gonorrhoeae</i> is more similar to that of <i>S. pneumoniae</i> and differs from that of <i>E. coli</i>. The comparisons revealed that compared to the interactions in <i>E. coli</i> and <i>S. pneumoniae</i>, more interactions between divisome proteins upstream of FtsA_Ng (including FtsA_Ng) and downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i> while fewer interactions between divisome proteins downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i>. Possible reasons for this include the inability of ZipA_Ng to interact with other divisome proteins and the absence of FtsL and FtsB in <i>N. gonorrhoeae</i>, resulting in the lack of an FtsQ-FtsB-FtsL complex in <i>N. gonorrhoeae</i>. These results indicate a possibly different divisome assembly in <i>N. gonorrhoeae</i> from that proposed models for <i>E. coli</i>.</p> A model for FtsA_Ng structure was predicted based on structural homology modeling with the resolved crystal structure of <i>Thermotoga maritima</i> FtsA. Four domains on the molecule were identified, designated 1A, 1C, 2B and 2A (including 2A₁ and 2A₂). Domains 2A and 2B of FtsA were highly conserved based on multi-sequence alignments of FtsAs from 30 bacteria. FtsA_Ng located to the division site in <i>N. gonorrhoeae</i> cells and the ratio of FtsA to FtsZ ranged from 1:24 to 1: 33 in three <i>N. gonorrhoeae</i> strains, which gave a lower cellular concentration of FtsA compared to other organisms.</p> <p>I also determined that overexpression of FtsA_Ng in <i>E. coli</i> led to cell filamentous in rod-shaped <i>E. coli</i> and cell enlargement and aggregation in mutant, round <i>E. coli</i>. FtsA_Ng failed to complement an <i>ftsA</i>_Ec-deletion <i>E. coli</i> strain although the overexperssion of FtsA_Ng disrupted <i>E. coli</i> cell division. In addition, overexpression of FtsA_Ng only affected cell division in some cells and its localization in <i>E. coli</i> was independent of interaction with <i>E. coli</i> FtsA or FtsZ. These results indicate that FtsA_Ng exhibits a species-specific functionality and <i>E. coli</i> is not a suitable model for studying FtsA_Ng functionality.</p> <p>This is the first study to characterize FtsA from <i>N. gonorrhoeae</i> in cell division. I identified novel functional domains of FtsA_Ng involved in interactions with other divisome proteins. The <i>N. gonorrhoeae</i> divisome protein interaction network determined by B2H assays provides insight into divisome assembly in <i>N. gonorrhoeae</i></p>.

In Arabidopsis, the F-box gene family encodes a large number of proteins postulated to act as substrate selectors for proteasome-mediated protein degradation. Recent reports document the importance of F-box proteins in developmental and metabolic signaling. Our microarray analyses of inflorescences of the brevipedicellus(bp) mutant indicate several F-box proteins are upregulated, suggesting that BP represses these genes in wild type plants to condition normal inflorescence development. We undertook analyses to examine the function of these proteins and their contribution to the pleiotropic phenotypes of bp. Yeast-2-hybrid screens revealed that the F-box protein At1g80440 binds to phenylalanine ammonia lyase-1(PAL1), the gateway enzyme of phenylpropanoid metabolism. Transgenic lines driven by the 35S cauliflower mosaic virus were attained but could not be propagated, suggesting a fatal phenotype. BP driven F-box expression results in phyllotaxy defects, manifest as alterations in the emergence of inflorescence and floral meristems in the axils of some cauline leaves.

The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC) transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22 interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique interactors belong to a wide variety of functional categories. There were 11 interactors involved in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4 interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2 interactors involved in stress response, and 1 interactor in each of the following categories: cell wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p play an important role in zinc homeostasis because deletion of these ABC transporters results in sensitivity to zinc shock.

The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC) transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22 interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique interactors belong to a wide variety of functional categories. There were 11 interactors involved in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4 interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2 interactors involved in stress response, and 1 interactor in each of the following categories: cell wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p play an important role in zinc homeostasis because deletion of these ABC transporters results in sensitivity to zinc shock.