In this study, the aim was to achieve high concentration of recombinant human growth hormone (rhGH) production by recombinant Pichia pastoris by investigating the effects of various operation parameters and to determine the suitable peptide ligand sequence that shows affinity and specificity to hGH. In this context, firstly the effect of temperature and Tween-20/80 addition on production and cell growth were investigated. While at T=30 and 32&deg / C, there was no difference, at 27 and 25&deg / C cell growth slowed down and production decreased significantly. The addition of Tween-20/80 in existence of co-substrate sorbitol did not affect the bioprocess while in absence of sorbitol Tween alone did not show the same positive effect on product formation and cell growth. Thereafter at T=30&deg / C, without addition of Tween, three sets of pilot scale bioreactor experiments were performed. In the first set, the effect of methanol feeding rate on bioprocess characteristics were investigated at the specific growth rates of &mu / =0.02, 0.03 and 0.04 h-1. While the highest cell concentration was achieved at &mu / =0.04 h-1, the highest rhGH concentration was achieved at &mu / =0.03 h-1. Secondly, conducting methanol feeding at &mu / =0.03 h-1, pH=5.5 experiment was conducted. The highest cell concentration, 45 g L-1 and maximum rhGH concentration 0.25 g L-1 were achieved at t=18 h of the process. Finally, the effect of batch sorbitol feeding on bioprocess was observed by the addition of 50 g L-1 sorbitol at t=0, 14 and 31 h of the production phase. It was shown that sorbitol addition to the medium increased process duration / hence cells enter stationary phase after a longer production phase. However, the protease concentration continued increasing with respect to time and at the end of the process reached twice the concentration it was obtained with single sorbitol addition case decreasing the rhGH concentration. In selection of the peptide sequence that shows affinity towards hGH, phage display method was conducted. Additionally the sequences from literature and computational design were used as alternatives. The interaction between these peptides and hGH was investigated by isothermal titration calorimetry and surface plasmon resonance.

Mycoplasma hyopneumoniae is the etiologic agent causing chronic pneumonia of swine. The lung lesions of swine produce the slower growth rate and lower feed conversion ratio and finally cause economic loss. Although four genome projects of mycoplasma species had been completed, the genome-sequencing project of M. hyopneumoniae also closed to the finished stage. However, only a few genes and proteins of M. hyopneumoniae have been studied, the molecular pathogenic mechanism remains elusive. The research of molecular vaccine is still preliminary. In order to obtain more information about epitope structures as the basis to develop molecular vaccine against this pathogen, two phage-displayed random heptapeptides libraries were used to identify epitopes recognized by purified IgG of rabbit anti-M. hyopneumoniae hyperimmune serum in this study. Individual phage clones were isolated and verified the binding specificity to the purified IgG by Western blot analysis and competitive ELISA after three rounds of biopanning. The selected clones were further characterized by DNA sequencing analysis and deduced to amino acid sequences. There are six consensus sequences contained tri- to hepta-peptide existing among the selected phage clones by aligning the sequences of foreign amino acids displaying on pIII protein. The consensus sequences may be serving as crucial epitopes of M. hyopneumoniae. By searching the protein database of M. hyopneumoniae deposited in NCBI, some surface proteins were matched by the selected mimotopes. Like P97, the essential protein for attaching to cilia of swine, the deduced epitopes mainly located at a.a. from 365 to 382, 395 to 403 and 436 to 452, the R1 and R2 repeated sequences also matched by the mimotopes. To evaluate the potential of these mimotopes as effective vaccine, several phage clones were chosen to immunize mice by intraperitoneal and intranasal administration. There are specific antibody responses to these mimotopes existing in serum IgG, fecal extracts and bronchoalveolar lavage fluids IgA. The serum IgG subclass profiles analysis reveals that these are mainly existed in IgG1 subclass. Base on the results of IgG subclass profiles analysis in sera, the results suggest that the phage-derived vaccines mainly activate Th2 cellular immunity pathway with the strategy used in this study. The similar results were found in the inactivated vaccine. The Th2 activation will promote the elimination of extracellular microorganism. Western blotting analysis showed that each serum raised by the phage clones could recognize 2 to 5 mycoplasma proteins. With the results of growth inhibition assay, we found that the selected phage clones CS4 and 58 are better vaccine candidates and some proteins (97 kDa¡B56 kDa and 30 kDa) may play crucial roles in block the bacteria growth. The advantage was taken of the natural property of M13 phage to infect E. coli, which is initiated by the N terminal of pIII coat protein binding with the F pili of E. coli. Plaque reduction tests were proposed to demonstrate the humoral immunity responses induced by phage-derived vaccine containing the antibodies specifically against the foreign peptide displayed on pIII coat protein. The present results provide more epitope information of M. hyopneumoniae. The mice immunization results reveal that the phage-displayed mimotopes can be used as potential vaccine candidates. The strategy presented in this study can shorten the time course for vaccine development and provide an alternative pathway for searching vaccine candidates against M. hyopneumoniae.

<p>The recent development of methods for constructing andhandling large collections (libraries) of proteins, from whichvariants with desired traits can be isolated, hasrevolutionized the field of protein engineering. Key elementsof such methods are the various ways in which the genotypes(the genes) and the phenotypes (the encoded proteins) arephysically linked during the process. In one section of thework underlying this thesis, one such technique (phagedisplay), was used to isolateand identify protein librarymembers based on their catalytic or target molecule-bindingproperties.</p><p>In a first study, phage display libraries of the lipolyticenzyme Lipolase from Thermomyces lanuginosa were constructed,the objective being to identify variants with improvedcatalytic efficiency in the presence of detergents. Toconstruct the libraries, nine positions were targeted for codonrandomization, all of which are thought to be involved in theconformational change-dependent enzyme activation that occursat water-lipid interfaces. The aim was to introduce two tothree amino acid mutations at these positions per lipase gene.After confirming that the wt enzyme could be functionallydisplayed on phage, selections with the library were performedutilizing a mechanism-based biotinylated inhibitor in thepresence of a detergent formulation. According to rhodamineB-based activity assays, the fraction of active clonesincreased from 0.2 to 90 % over three rounds of selection.Although none of the variants selected using this approachshowed increased activity, in either the presence or absence ofdetergent compared to the wild type enzyme, the resultsdemonstrated the possibility of selecting variants of theenzyme based on catalytic activity.</p><p>In the following work, phage libraries of the StaphylococcalProtein A (SPA)-derived Z-domain, constructed by randomizationof 13 surface-located positions, were used to isolate Z domainvariants (affibodies) with novel binding specificities. Astargets for selections, the parental SPA domains as well as twopreviously selected affibodies directed against two unrelatedtarget proteins were used. Binders of all three targets wereisolated with affinities (KD) in the range of 2-0.5 µM.One SPA binding affibody (Z_SPA-1) was shown to bind to each of the fivehomologous native IgG-binding domains of SPA, as well as theZdomain used as the scaffold for library constructions.Furthermore, the Z_SPA-1affibody was shown to compete with one of thenative domains of SPA for binding to the Fc part of humanantibodies, suggesting that the Z_SPA-1affibody bound to the Fc-binding surface ofthe Z domain. The majority of the affibodies isolated in theother two selections using two different affibodies as targets,showed very little or no binding to unrelated affibodies,indicating that the binding was directed to the randomizedsurface of their respective targets, analogously toanti-idiotypic antibodies.</p><p>The structure of the wild type Z domain/Z_SPA-1affibody co-complex was determined by x-raycrystallography, which confirmed the earlier findings in thatthe affibody Z_SPA-1affibody was shown to bind to the Fc bindingsurface of the Z domain. Further, both the Z domain and the Z_SPA-1affibody had very similar three helix-bundletopologies, and the interaction surface involved ten out of thethirteen randomized residues, with a central hydrophobic patchsurrounded by polar residues. In addition, the interactionsurface showed a surprisingly high shape complementarity, giventhe limited size of the library used for selections. The Z_SPA-1affibody was further investigated for use invarious biotechnological applications. In one study, the Z_SPA-1affibody was successfully recruited as a novelaffinity gene fusion partner for production, purification anddetection of cDNA-encoded recombinant proteins using anSPA-based medium for affinity chromatography. Further, the SPAbinding capability of the Z_SPA-1affibody was employed for site-specific andreversible docking of Z_SPA-1affibody-tagged reporter proteins onto an SPAfusion protein anchored to a cellulose surface via acellulose-binding moiety. These generated protein complexesresembles the architecture of so-called cellulosomes observedin cellulolytic bacteria. The results suggest it may bepossible to use anti-idiotypic affibody-binding protein pairsas modules to build other self-assembling types of proteinnetworks.</p><p><b>Keywords:</b>phage display, selection, mechanism-basedinhibitor, affinity domains, crystal structure, Staphylococcusaureus protein A, affinity chromatography, anti-idiotypicbinding pairs, affibody, combinatorial, protein engineering,lipase, cellulosome, assembly.</p>

B and T cells comprise the two major arms of the adaptive immune response tasked with clearing and preventing infection; molecular recognition in these cells occurs through antibodies and T cell receptors (TCRs), respectively. Highly successful therapeutics, clinical diagnostics and laboratory tools have been engineered from fragments of these parent molecules. The binding specificity, affinity and biophysical characteristics of these fragments determine their potential applications and resulting efficacies. Thus engineering desired properties into antibody and TCR fragments is a major concern of the multi-billion dollar biopharmaceutical industry. Toward this goal, we (1) designed antibody specificity using a novel computational method, (2) engineered thermoresistant Fabs by phage-based selection and (3) modulated binding kinetics for a single-chain TCR. In the first study, de novo modeling was used to generate libraries of FLAG peptide-binding single-chain antibodies. Phage-based screening identified a dominant design, and activity was confirmed after conversion to soluble Fab format. Bioinformatics analysis revealed potential areas for design process improvement. We present the first experimental validation of this in silico design method, which can be used to guide future antibody specificity engineering efforts. In the second study, the variable heavy chain of a moderately stable EE peptide-binding Fab was subjected to random mutagenesis, and variants were selected for resistance to heat inactivation. Thermoresistant clones where biophysically characterized, and structural analysis of selected mutations suggested general mechanisms of stabilization. Framework mutations conferring thermoresistance can be grafted to other antibodies in future Fab stabilization work. In the third study, TCR fragment binding kinetics for a clonotypic antibody were modulated by varying valence during phage-based selection. Binding affinity and kinetics for representative variants depended on the display format used during selection, and all TCR fragments retained binding to native pMHC antigen. This work demonstrates a general engineering platform for tuning protein-protein interactions. Using a combination of computational design and phage-based screening, we have identified antibodies and TCR fragments with improved binding properties or biophysical characteristics. The optimized variants possess a wider range of potential applications compared to their parent molecules, and we detail engineering methods likely to be useful in the engineering of many other protein-based therapeutics. / text

The conflict between HIV-1 and the host immune system plays out over a time-scale of months and years, and on a grander scale in the co-evolution of lentiviruses and the immune systems of their host species. Directed evolution of HIV-1 entry inhibitors using controlled randomization together with a display system offers a means of recapitulating one side of this conflict in vitro on an accelerated time-scale. To address limitations in existing display systems, we constructed a vector (pDQ1) integrating phage-display and mammalian-expression systems. This vector displays on phage when expressed in bacteria, and as an Fc-fusion when expressed in tissue culture, thus accelerating the iterative process of randomization, display, and characterization. We demonstrated the utility of this vector in the evolution of a CD4-mimetic peptide.

This dissertation summarizes advances made toward understanding of the composition, structure, mechanism, and regulation of the bacterial type VI secretion system (T6SS). The T6SS is a widely conserved bacterial nanomachine used by Gram-negative bacteria to deliver toxic effector proteins into the extracellular environment or into adjacent target cells. Systematic deletion of open reading frames present in the Vibrio cholerae T6SS gene cluster revealed the genes essential for T6SS activity and provided key insights into understanding the mechanism by which this organelle is assembled and its components are recycled. Characterization of one phage-related T6SS component yielded insight into the mechanism by which many effectors associate with the T6SS organelle and are delivered into target cells. This T6SS component serves both to sharpen the tip of the membrane-puncturing T6SS spike complex and as a vehicle for attaching a diverse set of effector proteins. Time-lapse fluorescence microscopy of GFP-labeled T6SS components revealed key insights into the behavior and regulation of the T6SS in Pseudomonas aeruginosa. The T6SS in this organism assembled in response to exogenous T6SS attack by adjacent sister cells as well as heterologous T6SS+ species V. cholerae and Acinetobacter baylyi. This retaliatory T6SS counterattack was precisely aimed and caused no collateral damage to neighboring, non-aggressive bacteria. These counterattacks are mediated by phosphorylation cascade that recognizes exogenous attacks and post-translationally activates the T6SS in P. aeruginosa. Deletion of genes in this pathway eliminated the retaliatory response while retaining T6SS functionality. This pathway also induced T6SS counterattacks in response to mating pair formation associated with type IV secretion system (T4SS)-mediated DNA conjugation as well as treatment with membrane-disrupting natural product polymyxin B, suggesting that the signal needed to induce T6SS activity was mechanical perturbation of the P. aeruginosa cell membrane. Interestingly, these T4SS-induced counterattacks were able to confer resistance to the acquisition of horizontally transferred foreign DNA by selectively killing conjugative donor cells. As such, the T6SS of P. aeruginosa may represent a type of general bacterial innate immune system capable of responding to a wide range of exogenous threats.

Viruses infecting bacteria (phages) are the most abundant and ubiquitous entities on Earth and likely critical to any ecosystem, as they influence nutrient cycling, mortality and evolution. Ultimately, their impact depends on whether phage—host interactions lead to intracellular phage coexistence (temperate phage) or cell death (lytic phage). Temperate phages in the lysogenic cycle replicate their genome (either integrated into the host chromosome or extrachromosomally), until induced to become lytic, when they create and release progeny via cell lysis. While knowledge on lytic versus lysogenic outcomes is vast, it largely derives from few model systems that underrepresent natural diversity. Further, less is known about the efficiency of phage—host interactions and the regulation of optimal versus sub-optimal lytic infections, which are predicted as relevant under environmental (nutrients, temperature) and host (availability, density) conditions that are common in the ocean. In this dissertation I characterize the phage—host interactions in a new marine model system, phage ϕ38:1 and its Cellulophaga baltica bacterial host, member of the ubiquitous Bacteroidetes phylum. First, I show ϕ38:1’s ability to infect numerous, genetically similar strains of the C. baltica species, two of which display contrasting infection outcomes–lytic versus sub-optimally lytic or lysogenic on the original versus alternative hosts, respectively. Second, I collaboratively apply new gene marker-based approaches (phageFISH and geneELISA) to study ϕ38:1’s infection at the single-cell level and show that it is sub-optimal on the alternative host, rather than lysogenic. Third, I collaboratively develop whole-genome transcriptome datasets for ϕ38:1 infecting both, the optimal and sub-optimal hosts, to characterize the cellular response to infection and hypothesize potential transcriptional and post-transcriptional regulation of the sub-optimal infection. Together, these findings advance our knowledge of naturally-occurring phage—host interactions with a focus on nearly-unstudied sub-optimal infections.

No description available.

Applications of biotechnology in drug delivery and medical instrumentation and energy storage have been gaining popularity. Especially, utilization of biotechnology for energy storage is attracting attention due to its environmentally friendly nature and cost efficiency. In this project, a filamentous bacteriophage, M13, to fabricate metal oxide battery electrodes. M13 phage is 6.5 nm wide and 800 nm long, and can act as a template to produce nano-sized metal oxide particles. A method to prepare manganese oxide electrodes was developed, where the phage is integrated with the oxide into a nanocomposite. The composite material was used to make a high capacity electrode for lithium ion batteries. The M13 templated manganese oxide, Mn3O4, could deliver a high initial capacity of 766 mAh/g, and recorded a stabilized discharge capacity of ~800 mAh/g even after 60 cycles.

Mast cells are innate immune cells, historically involved in allergy responses involving IgE. Through this, they have earned a reputation as a fairly detrimental cell type. Their beneficial roles remain somewhat enigmatic although they clearly have the ability to modulate the immune system. This is due to their ability to synthesise many cytokines and chemokines as well as immediately release potent granule-stored mediators. One such mediator is a serine protease, chymase, which has been targeted by pharmaceutical companies developing inhibitors for use in inflammatory conditions. In order to address roles of the proteases, information regarding their cleavage specificity using substrate phage display can help find potential in vivo substrates.  The human chymase cleaves substrates with aromatic amino acids in the P1 position and has a preference for negatively charged amino acids in the P2’ position. The molecular interactions mediating this P2’ preference was investigated by site-directed mutagenesis, where Arg143 and Lys192 had a clear effect in this selectivity. As humans express one chymase and rodents express multiple chymases, extrapolating data between species is difficult. Here, the crab-eating macaque was characterised, which showed many similarities to the human chymase including a near identical extended cleavage specificity and effects of human chymase inhibitors.  Appropriate models are needed when developing human inhibitors for therapeutic use in inflammatory conditions. The effects of five specific chymase inhibitors in development were also tested. The selectivity of inhibitors was dependent on both Arg143 and Lys192, with a greater effect of Lys192. Identification of residues involved in specific inhibitor interactions is important for selective inhibitor development. Another innate cell type, the NK cell, is important in virus and tumour defence. In the channel catfish, a serine protease from an NK-like cell, granzyme-like I, was characterised. A strict preference for Met in the P1 position was seen, and caspase 6 was identified as a potential in vivo target. This may highlight a novel apoptosis-inducing mechanism from a similar cell type has been conserved for approximately 400 myr. Here, important residues mediating chymases’ specificity and interactions with inhibitors has been addressed, as well as finding a new animal model for providing ways to combat their roles in pathological settings.