Unveiling the secrets of DNA : Improved expression and phage display efficiency of synthetic recombinant binding proteins in E. coli through modulation of codon usage

Recombinant binding proteins are becoming increasingly important in various applications, including diagnostics, basic research, and therapeutics. Therefore, the demand for recombinant binding proteins will increase in the future, making it essential to enhance protein production methods. One approach is to improve heterologous expression, in which proteins are produced outside their native hosts. scherichia coli has been a workhorse of heterologous expression for decades due to easy cultivation, cost efficiency, well-known genetics, and compatibility with phage display. However, the production of heterologous proteins in E. coli can sometimes be very difficult. Often the problems in heterologous expression are related to codon usage, which works as a control mechanism of protein translation, especially in bacteria. Different organisms do not use codons in the same manner, and in some cases, a heterologously expressed gene can include codons that are rarely or too frequently used in E. coli, which can disturb the codon usage derived control of translation. Many studies have reported improved yields of heterologous proteins produced in E.coli when the codon usage of the heterologous gene has been recoded to suit better for E. coli. Most often, recombinant binding proteins used in the previously mentioned applications are not native proteins of E. coli either, and many times their production or phage display in E. coli can be very cumbersome. In this thesis, we aimed to improve expression and phage display properties of two essential recombinant binding proteins in E. coli through modulation of codon usage. One binding protein was an antibody fragment called fragment antigen binding (Fab) and another artificial recombinant binding protein called Designed Ankyrin Repeat Protein (DARPin). In the first publication, the expression and phage display efficiency of a human anti-digoxigenin Fab fragment was improved by codon harmonizing the selected segments of the Fab fragment gene. In the second publication, expression and secretion properties of Fab fragments were improved by selecting enhanced signal sequence variants from PelB signal sequence libraries, which included only codon changes. In the third publication, the signal sequence libraries were used to enhance Sec dependent phage display of an anti-GFP DARPin and a DARPin library

Tuotannon ja T&K-toiminnan integraatio

Tuotannon ja T&K-toiminnan tehtävät ja tavoitteet eroavat usein toisistaan. Näiden välisellä integraatiolla tarkoitetaan sellaista yhteistyötä, jossa molemmat toiminnot hyötyvät yhteisistä toimintakäytänteistä hyödyntäen yhteisiä informaatiojärjestelmiä. Tällainen yhteistyö auttaakin yhteensovittamaan näiden tehtäviä ja tavoitteita pyrkien yrityksen kokonaistavoitteeseen välttäen toimintojen osaoptimointia. Integraatio luo yritykselle mahdollisuuden parantaa kilpailukykyään keskeisimmissä kilpailukykytekijöissä eli kustannuksissa, innovatiivisuudessa, joustavuudessa, laadussa ja T&K-toiminnan suorituskyvyssä. Aihe onkin merkityksellinen, sillä se koskettaa suurinta osaa teollisuusyrityksistä. Tämän tutkimuksen tarkoituksena on selvittää, mitä tuotannon ja T&K-toiminnan integraatiolla voidaan saavuttaa, ja missä tapauksissa näiden toimintojen läheinen yhteistyö on tarpeen. Tavoitteena on kuvata ne integraation avaintekijät, jotka ovat keskeisiä synergiaetujen saavuttamisessa. Tämä työ tarkasteleekin synergiaetuja jokaisen kilpailukykytekijän näkökulmasta sekä eri avaintekijöihin keskittyen. Tämä tutkimus toteutettiin kirjallisuustutkielmana, jonka aineiston keruu ja analysointi toteutettiin systemaattisesti tutkimuskysymyksiin vastaamiseksi. Työ jakautuu seuraavalla tavalla kolmeen lukuun: Luvussa 1 kuvataan tutkimuksen tavoitteet, tutkimusmenetelmät ja työn tarkempi rakenne. Luvussa 2 keskitytään tuotannon ja T&K-toiminnan synergiaetuihin jokaisen kilpailukykytekijän osalta kiertotalous huomioiden. Luvussa 3 käsitellään tuotannon ja T&K-toiminnan integraation avaintekijöitä sijaintipäätösten, keskittämistarpeen ja globaalin toimintaympäristön näkökulmasta. Lisäksi luvussa 3 kuvataan poikkitoiminnallisen integraation, osallistavan organisaatiokulttuurin, integraation tuotetasolla tuotesuunnittelun ja prosessien eri vaiheissa sekä toiminnanohjausjärjestelmien merkitys integraatiossa. Luvussa 4 luodaan synteesi lukujen 2 ja 3 pohjalta. Työn lopussa esitetään aineiston pohjalta yhteenveto tuloksista sekä näiden perusteella syntyneet päätelmät. Tulosten ja päätelmien pohjalta voidaan todeta, että tuotannon ja T&K-toiminnan avulla voidaan saavuttaa synergiaetuja jokaisessa kilpailukykytekijässä eli joustavuudessa, laadussa, innovatiivisuudessa, kustannuksissa ja T&K-toiminnan suorituskyvyssä. Tuotanto ja T&K-toiminta vaativat läheistä yhteistyötä tietointensiivisyyden eli tuotteiden ja prosessien kompleksisuuden ollessa korkeaa. Nämä toiminnot tulee sijaita maantieteellisesti samassa paikassa T&K-toiminnan keskittyessä suoraan tuotannon prosessiteknologian kehittämiseen uusien tuotteiden sijaan. Integraatio toteutetaan tehokkaasti huomioimalla avaintekijät eli sijaintipäätökset, poikkitoiminnallinen integraatio, osallistava organisaatiokulttuuri, integraatio tuotetasolla kehitysprosessin eri vaiheissa ja toiminnanohjausjärjestelmät. Tämä työ tarjoaakin arvokasta tietoa tuotannon ja T&K-toiminnan integraatiopäätösten tueksi yrityksen johdolle

Coalitional Game Theory based Value Sharing in Energy Communities

This paper presents a coalitional game for value sharing in energy communities (ECs). It is proved that the game is super-additive, and the grand coalition effectively increases the global payoff. It is also proved that the model is balanced and thus, it has a nonempty core. This means there always exists at least one value sharing mechanism that makes the grand coalition stable. Therefore, prosumers will always achieve lower bills if they join to form larger ECs. A counterexample is presented to demonstrate that the game is not convex and value sharing based on Shapley values does not necessarily ensure the stability of the coalition. To find a stabilizing value sharing mechanism that belongs to the core of the game, the worst-case excess minimization concept is applied. In this concept, however, size of the optimization problem increases exponentially with respect to the number of members in EC. To make the problem computationally tractable, the idea of clustering members based on their generation/load profiles and considering the same profile and share for members in the same cluster is proposed here. K-means algorithm is used for clustering prosumers’ profiles. This way, the problem would have several redundant constraints that can be removed. The redundant constraints are identified and removed via the generalized Llewellyn’s rules. Finally, value sharing in an apartment building in the southern part of Finland in the metropolitan area is studied to demonstrate effectiveness of the method

Finnish agriculture and rural industries 2007

The report consists of following topics: operating environment of agriculture, agricultural and food market, agricultural policy, economic situation of agriculture, agriculture and the environment, rural and regional policy, special topics: biogas etc

Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins

DsbA leader peptide targets proteins for cotransla-tional translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44-and 12-fold compared to PelBWT and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelBWT, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency

Coordination concepts for interactions between energy communities, markets and distribution grids

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The effect of cloudiness on new-particle formation : investigation of radiation levels

Peer reviewe

Soil concentrations and soil-atmosphere exchange of alkylamines in a boreal Scots pine forest

Alkylamines are important precursors in secondary aerosol formation in the boreal forest atmosphere. To better understand the behavior and sources of two alkylamines, dimethylamine (DMA) and diethylamine (DEA), we estimated the magnitudes of soil-atmosphere fluxes of DMA and DEA using a gradient-diffusion approximation based on measured concentrations in soil solution and in the canopy air space. The ambient air concentration of DMA used in this study was a sum of DMA and ethylamine. To compute the amine fluxes, we first estimated the soil air space concentration from the measured soil solution amine concentration using soil physical (temperature, soil water content) and chemical (pH) state variables. Then, we used the resistance analogy to account for gas transport mechanisms in the soil, soil boundary layer, and canopy air space. The resulting flux estimates revealed that the boreal forest soil with a typical long-term mean pH 5.3 is a possible source of DMA (170 +/- 51 nmolm(-2) day(-1)) and a sink of DEA (-1.2 +/- 1.2 nmolm(-2) day(-1)). We also investigated the potential role of fungi as a reservoir for alkylamines in boreal forest soil. We found high DMA and DEA concentrations both in fungal hyphae collected from field humus samples and in fungal pure cultures. The highest DMA and DEA concentrations were found in fungal strains belonging to decay and ectomycorrhizal fungal groups, indicating that boreal forest soil and, in particular, fungal biomass may be important reservoirs for these alkylamines.Peer reviewe

Evaluating CENTURY and Yasso soil carbon models for CO2 emissions and organic carbon stocks of boreal forest soil with Bayesian multi-model inference

We can curb climate change by improved management decisions for the most important terrestrial carbon pool, soil organic carbon stock (SOC). However, we need to be confident we can obtain the correct representation of the simultanous effect of the input of plant litter, soil temperature and water (which could be altered by climate or management) on the decomposition of soil organic matter. In this research, we used regression and Bayesian statistics for testing process-based models (Yasso07, Yasso15 and CENTURY) with soil heterotrophic respiration (Rh) and SOC, measured at four sites in Finland during 2015 and 2016. We extracted climate modifiers for calibration with Rh. The Rh values of Yasso07, Yasso15 and CENTURY models estimated with default parameterization correlated with measured monthly heterotrophic respiration. Despite a significant correlation, models on average underestimated measured soil respiration by 43%. After the Bayesian calibration, the fitted climate modifier of the Yasso07 model outperformed the Yasso15 and CENTURY models. The Yasso07 model had smaller residual mean square errors and temperature and water functions with fewer, thus more efficient, parameters than the other models. After calibration, there was a small overestimate of Rh by the models that used monotonic moisture functions and a small generic underestimate in autumn. The mismatch between measured and modelled Rh indicates that the Yasso and CENTURY models should be improved by adjusting climate modifiers of decomposition or by accounting for missing controls in, for example, microbial growth.Peer reviewe