Design and production of protein nanostructures for biomolecular detection

Particulate nanostructures are increasingly used for analytical purposes. Such particles are often generated by chemical synthesis from non-renewable raw materials. Generation of uniform nanoscale particles is challenging and particle surfaces must be modified to make the particles biocompatible and water-soluble. Usually nanoparticles are functionalized with binding molecules (e.g., antibodies or their fragments) and a label substance (if needed). Overall, producing nanoparticles for use in bioaffinity assays is a multistep process requiring several manufacturing and purification steps. This study describes a biological method of generating functionalized protein-based nanoparticles with specific binding activity on the particle surface and label activity inside the particles. Traditional chemical bioconjugation of the particle and specific binding molecules is replaced with genetic fusion of the binding molecule gene and particle backbone gene. The entity of the particle shell and binding moieties are synthesized from generic raw materials by bacteria, and fermentation is combined with a simple purification method based on inclusion bodies. The label activity is introduced during the purification. The process results in particles that are ready-to-use as reagents in bioaffinity. Apoferritin was used as particle body and the system was demonstrated using three different binding moieties: a small protein, a peptide and a single chain Fv antibody fragment that represents a complex protein including disulfide bridge.If needed, Eu3+ was used as label substance. The results showed that production system resulted in pure protein preparations, and the particles were of homogeneous size when visualized with transmission electron microscopy. Passively introduced label was stably associated with the particles, and binding molecules genetically fused to the particle specifically bound target molecules. Functionality of the particles in bioaffinity assays were successfully demonstrated with two types of assays; as labels and in particle-enhanced agglutination assay. This biological production procedure features many advantages that make the process especially suited for applications that have frequent and recurring requirements for homogeneous functional particles. The production process of ready, functional and watersoluble particles follows principles of “green chemistry”, is upscalable, fast and cost-effective.

Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.

Phylogenetic Analysis of Avidins and Expression of Novel Avidins from Lactrodectus hesperus and Hoeflea phototrophica

Avidins (Avds) are homotetrameric or homodimeric glycoproteins with typically less than 130 amino acid residues per monomer. They form a highly stable, non-covalent complex with biotin (vitamin H) with Kd = 10-15 M (for chicken Avd). The best-studied Avds are the chicken Avd from Gallus gallus and streptavidin from Streptomyces avidinii, although other Avd studies have also included Avds from various origins, e.g., from frogs, fishes, mushrooms and from many different bacteria. Several engineered Avds have been reported as well, e.g., dual-chain Avds (dcAvds) and single-chain Avds (scAvds), circular permutants with up to four simultaneously modifiable ligand-binding sites. These engineered Avds along with the many native Avds have potential to be used in various nanobiotechnological applications. In this study, we made a structure-based alignment representing all currently available sequences of Avds and studied the evolutionary relationship of Avds using phylogenetic analysis. First, we created an initial multiple sequence alignment of Avds using 42 closely related sequences, guided by the known Avd crystal structures. Next, we searched for non-redundant Avd sequences from various online databases, including National Centre for Biotechnology Information and the Universal Protein Resource; the identified sequences were added to the initial alignment to expand it to a final alignment of 242 Avd sequences. The MEGA software package was used to create distance matrices and a phylogenetic tree. Bootstrap reproducibility of the tree was poor at multiple nodes and may reflect on several possible issues with the data: the sequence length compared is relatively short and, whereas some positions are highly conserved and functional, others can vary without impinging on the structure or the function, so there are few informative sites; it may be that periods of rapid duplication have led to paralogs and that the differences among them are within the error limit of the data; and there may be other yet unknown reasons. Principle component analysis applied to alternative distance data did segregate the major groups, and success is likely due to the multivariate consideration of all the information. Furthermore, based on our extensive alignment and phylogenetic analysis, we expressed two novel Avds, lacavidin from Lactrodectus Hesperus, a western black widow spider, and hoefavidin from Hoeflea phototrophica, an aerobic marine bacterium, the ultimate aim being to determine their X-ray structures. These Avds were selected because of their unique sequences: lacavidin has an N-terminal Avd-like domain but a long C-terminal overhang, whereas hoefavidin was thought to be a dimeric Avd. Both these Avds could be used as novel scaffolds in biotechnological applications.

Strategic supply chain alignment to customer buying behavior

In search for competitive advantage, designing and managing supply chain networks have become a necessary competence for organizations. The target of this thesis is to answer a question, how to design a multiple supply chain network. The purpose is to study, what kind of different supply chain designs exist and, how to choose appropriate supply chain designs for a company. In the thesis, the focus is on the supply chain alignment to customers, more specifically to customer buying behavior. The research method was a case study. A framework for measuring customer buying behavior was developed based on the literature and it was used in the study of customer buying behavior in the case environment. In the case company structured interviews and data records were used as sources of evidence. Persons working in the customer-interface were interviewed face-to-face and through an e-mail questionnaire. When analyzing the data, a Quality function deployment matrix was used as one analysis method. As a result of the thesis, supply chain network of the case company is proposed to be divided into three separate supply chains, which focus on different areas and they could be called lean, agile and continuous replenishment supply chains. In conclusion, in the supply chain alignment to customer buying behavior several aspects have to be studied from different perspectives. According to the results, a multiple supply chain strategy is recommended to be implemented in the case company, since the diversity of the customer needs cannot be managed efficiently through a single supply chain.

The demand-supply balancing process in supply chain management - case mining & metals industry equipment

The aim of this thesis is to search how to match the demand and supply effectively in industrial and project-oriented business environment. The demand-supply balancing process is searched through three different phases: the demand planning and forecasting, synchronization of demand and supply and measurement of the results. The thesis contains a single case study that has been implemented in a company called Outotec. In the case study the demand is planned and forecasted with qualitative (judgmental) forecasting method. The quantitative forecasting methods are searched further to support the demand forecast and long term planning. The sales and operations planning process is used in the synchronization of the demand and supply. The demand forecast is applied in the management of a supply chain of critical unit of elemental analyzer. Different meters on operational and strategic level are proposed for the measurement of performance.