<p>DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. </p><p>To expand the knowledge of protein–DNA recognition, we have utilized<i> in vitro</i> evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. </p><p>Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring <i>K</i>_D values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure.</p><p>mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. </p><p>The two <i>in vitro</i> selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.</p>

This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting. In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a Toxoplasma gondii surface antigen through hydrophobic interaction, lipids were added either in vivo via E. coli expression, or in vitro via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a Neospora caninum surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines. In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His6-ZHER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His6-(ZHER2/neu:4)2, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.

It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease. The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (&lt;6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma.

DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. To expand the knowledge of protein–DNA recognition, we have utilized in vitro evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring KD values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure. mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. The two in vitro selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.

Mast cells (MC) are potent inflammatory cells that are known primarily for their prominent role in IgE mediated allergies. However, they also provide beneficial functions to the host, e.g. in bacterial and parasitic defence. MCs react rapidly upon stimulation by releasing potent granule-stored mediators, and serine proteases of the chymase or tryptase families are such major granule constituents. As a first step towards a better understanding of the biological function of these proteases, we have determined the extended cleavage specificities of four mammalian mast cell chymases, by utilizing a substrate phage display approach. The specificities of these enzymes have then been used to compare their functional characteristics. The major mucosal MC chymase in mice, mMCP-1, was found to possess a strict preference in four amino acid positions of the peptide substrate. Using this sequence to search the mouse proteome for potential in vivo substrates led to the identification of several very interesting potential novel substrates. Some of them may explain the increased epithelial permeability provided by this enzyme. Human MCs, express only one single α-chymase, and the rodent α-chymases have secondarily gained elastase-like primary cleavage specificity. However, rodents express additional chymases, the β-chymases, and rodent β-chymases may have adopted the function of the α-chymases. The cleavage specificities of the human chymase and two rodent β-chymases were therefore determined (rat rMCP-1 and mouse mMCP-4). N-terminal of the cleaved bond the three chymases showed similar preferences, but C-terminal the human chymase and mMCP-4 shared a high preference for acidic amino acids in the P2´ position and therefore seem to be functional homologues. The molecular interactions mediating the preference for acidic amino acids in position P2´ were further investigated. By site-directed mutagenesis of the human chymase, amino acids Arg143 and Lys192 were concluded to synergistically mediate this preference. Our data show that chymases, of different MC subpopulations, display quite different extended cleavage specificities. However mouse do possess a MC chymase with almost identical cleavage specificity as the human MC chymase indicating a strong evolutionary pressure to maintain this enzyme specificity.

This thesis describes generation and characterization of Affibody molecules with future applications in proteomics research, protein structure determinations, therapeutic treatment of disease and medical imaging for in vivo diagnostics. Affibody molecules are engineered affinity proteins developed by combinatorial protein engineering from the 58-residue protein A-derived Z domain scaffold. Novel Affibody molecules targeting human proteins were selected from a combinatorial library using phage display technology. In the first two investigations, an Affibody molecule specifically targeting the high abundant human serum protein transferrin was generated. The intended future use of this Affibody ligand would be as capture ligand for depletion of transferrin from human samples in proteomics analysis. Strong and highly specific transferrin binding of the selected Affibody molecule was demonstrated by biosensor technology, dot blot analysis and affinity chromatography. Efficient Affibody-mediated depletion of transferrin in human plasma and cerebrospinal fluid (CSF) was demonstrated in combination with IgG and HSA removal. Furthermore, depletion of five high abundant proteins including transferrin from human CSF gave enhanced identification of proteins in a shotgun proteomics analysis. Two studies involved the selection and characterization of Affibody molecules recognizing Alzheimer’s amyloid beta (Abeta) peptides. Future prospect for the affinity ligands would primarily be for therapeutic applications in treatment of Alzheimer’s disease. The developed A-binding Affibody molecules were found to specifically bind to non-aggregated forms of Abeta and to be capable of efficiently and selectively capture Abeta peptides from spiked human serum. Interestingly, the Abeta-binding Affibody ligands were found to bind much better to Abeta as dimeric constructs, and with impressive affinity as cysteine-bridged dimers (KD~17 nM). NMR spectroscopy studies revealed that the original helix one, of the two Affibody molecules moieties of the cysteine-bridged dimers, was unfolded upon binding, forming intermolecular β-sheets that stabilized the Abeta peptide, enabling a high resolution structure of the peptide. Furthermore, the Abeta-binding Affibody molecules were found to inhibit Abeta fibrillation in vitro. In the last study, Affibody molecules directed to the interleukin 2 (IL-2) receptor alpha (CD25) were generated. CD25-binding Affibody molecules could potentially have a future use in medical imaging of inflammation, and possibly in therapeutic treatment of disease conditions with CD25 overexpression. The selected Affibody molecules were demonstrated to bind specifically to human CD25 with an apparent affinity of 130-240 nM. Moreover, the CD25-targeting Affibody molecules were found to have overlapping binding sites with the natural ligand IL-2 and an IL-2 blocking monoclonal antibody. Furthermore, the Affibody molecules demonstrated selective binding to CD25 expressing cells. / QC 20100729

Tumor targeting and molecular imaging of protein markers specific for or overexpressed in tumors can add useful information in deciding upon treatment and assessing the response to treatment for a cancer patient. The epidermal growth factor receptor (EGFR) is one such tumor-associated receptor, which expression is abnormal or upregulated in various cancers and associated with a poor patient prognosis. It is therefore considered a good target for imaging and therapy. Monoclonal antibodies and recently also antibody fragments have been investigated for in vivo medical applications, like therapy and imaging. In molecular imaging a small sized targeting agent is favorable to give high contrast and therefore, antibody fragments and lately also small affinity proteins based on a scaffold structure constitute promising alternatives to monoclonal antibodies. Affbody molecules are such affinity proteins that are developed by combinatorial protein engineering of the 58 amino acid residue Z-domain scaffold, derived from protein A. In this thesis, novel Affibody molecules specific for the EGFR have been selected from a combinatorial library using phage display technology. Affibody molecules with moderate high affinity demonstrated specific binding to native EGFR on the EGFR-expressing epithelial carcinoma A431 cell line. Further cellular assays showed that the EGFR-binding Affibody molecules could be labeled with radiohalogens or radiometals with preserved specific binding to EGFR-expressing cells. In vitro, the Affibody molecule demonstrated a high uptake and good retention to EGFR-expressing cells and was found to internalize. Furthermore, successful imaging of tumors in tumor-bearing mice was demonstrated. Low nanomolar or subnanomolar affinities are considered to be desired for successful molecular imaging and a directed evolution to increase the affinity was thus performed. This resulted in an approximately 30-fold improvement in affinity, yielding EGFR-binding Affibody molecules with KD´s in the 5-10 nM range, and successful targeting of A431 tumors in tumor-bearing mice. To find a suitable format and labeling, monomeric and dimeric forms of one affinity matured binder were labeled with 125I and 111In. The radiometal-labeled monomeric construct, 111In-labeled-ZEGFR:1907, was found to provide the best tumor-to-organ ratio due to good tumor localization and tumor retention. The tumor-to-blood ratio, which is often used as a measure of contrast, was 31±8 at 24 h post injection and the tumor was clearly visualized by gamma-camera imaging. Altogether, the EGFR-binding Affibody molecule is considered a promising candidate for further development of tumor imaging tracers for EGFR-expressing tumors and metastases. This could simplify the stratification of patients for treatment and the assessment of the response of treatment in patients. / QC 20100723

Protein-protein interactions are of vital importance to the cell as they mediate the assembly of protein complexes that carry out diverse biological functions. Many proteins involved in cellular signaling are built by the combinatorial use of peptide recognition modules (PRMs), which are small protein domains that bind to their cognate ligands by recognizing short linear peptide motifs. Thousands of PRMs are found in nature, requiring improved methods to better elucidate their molecular determinants of binding and to allow accurate mapping of their interaction networks. In this thesis, I describe the development and application of phage-displayed peptide libraries to map the binding specificities of two common PRMs. First, I generated specificity profiles for 82 C. elegans and human PDZ domains that could be organized into a specificity map. The map revealed that PDZ domains have far greater substrate sequence specificity than previously believed, providing significant insights into the relationships between PDZ structure and specificity, and allowing specificity prediction for uncharacterized domains. My results were used to predict both endogenous and pathogenic PDZ interactions. This analysis revealed that viruses have evolved ligands that specifically mimic PDZ domains to subvert host cell immunity. Second, I analyzed the binding specificity for the SH3 domain family in S. cerevisae. I found that, like PDZ domains, SH3 domains have binding specificities that are more detailed than the conventional classification system. The phage-derived specificity profiles were combined with data from oriented peptide and yeast two-hybrid screening to generate a highly accurate SH3 domain interaction network. Given the prominent role of SH3 domains in endocytosis, the SH3 domain interaction data was used to predict the dynamic localization of several uncharacterized endocytosis proteins, which was subsequently confirmed by cell-based assays. The application of the techniques described here to other PRM families will significantly improve protein interaction maps for signaling pathways, which will illuminate our understanding of the cell circuitry, allow the use of PRMs as general affinity reagent and detection tools, and guide the development of small molecule inhibitors that mimic their peptide ligands for therapeutic intervention.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Bacteriophage Lambda (λ) has played a historical role as an essential model contributing to our current understanding of molecular genetics. Lambda’s major capsid protein “gpD” occurs on each capsid at 405 to 420 copies per phage in homotrimeric form and functions to stabilize the head and likely to compact the genomic DNA. The interesting conformation of this protein allows for its exploitation through the genetic fusion of peptides or proteins to either the amino or carboxy terminal end of gpD, while retaining phage assembly functionality and viability. The lytic nature of λ and the conformation of gpD in capsid assembly makes this display system superior to other display options. Despite previous reports of λ as a phage display candidate, decorative control of the phage remains an elusive concept. The primary goal of this study was to design and construct a highly controllable head decoration system governed by two genetic conditional regulation systems; plasmid-mediated temperature sensitive repressor expression and bacterial conditional amber mutation suppression. The historical λ Dam15 conditional allele results in a truncated gpD fragment when translated in nonsuppressor, wild-type E. coli cells, resulting in unassembled, nonviable progeny. I sequenced the Dam15 allele, identifying an amber (UAG) translational stop at the 68th codon. Employing this mutant in combination with a newly created isogenic cellular background utilizing the amber suppressors SupD (Serine), SupE (Glutamine), SupF (Tyrosine) and Sup— (wild type), we sought to control the level of incorporation of undecorated gpD products. As a second dimension, I constructed two separate temperature-inducile plasmids whereby expression of either D or D::eGFP was governed by the λ strong λ CI[Ts]857 temperature-sensitive repressor and expressed from the λ PL strong promoter. Our aim was to measure the decoration of the λ capsid by a D::gfp fusion under varying conditions regulated by both temperature and presence of suppression. This was achieved utilizing this controllable system, enabling the measurement of a variable number of fusions per phage based on diverse genetic and physical environments without significantly compromising phage viability. Surprisingly, both SupE and SupF showed similar levels of Dam15 suppression, even though sequencing data indicated that only SupE could restore the native gpD sequence at amino acid 68 (Q). In contrast, SupD (S), conferred very weak levels of suppression, but imparted an environment for very high decoration of gpD::eGFP per capsid, even at lower (repressed) temperatures. The presence of albeit few wild-type gpD molecules allowed for an even greater display than that of the perceived “100%” decoration scenario provided by the nonsuppressor strain. It appears that the lack of wild-type gpD does not allow for the space required to display the maximum number of fusions and in turn creates an environment that affects both phage assembly and therefore phage viability. Finally, the use of Western blotting, confirmed the presence of gpD::eGFP fusion decoration by employing a polyclonal anti-eGFP antibody. The significance of this work relates to the unique structure of λ’s capsid and its ability to exploit gpD in the design of controlled expression, which is guiding future research examining the fusion of different therapeutic peptides and proteins. Furthermore this approach has important implications specifically for the design of novel vaccines and delivery vehicles for targeted gene therapy in which steric hindrance and avidity are important concerns. The execution of this project employed basic bacterial genetics, phage biology and molecular biology techniques in the construction of bacterial strains and plasmids and the characterization of the phage display system.