Evolution of Bioaffinity Reagents by Phage Display

Antibodies are natural binding proteins produced in vertebrates as a response to invading pathogens and foreign substances. Because of their capability for tight and specific binding, antibodies have found use as binding reagents in research and diagnostics. Properties of cloned recombinant antibodies can be further improved by means of in vitro evolution, combining mutagenesis with subsequent phage display selection. It is also possible to isolate entirely new antibodies from vast naïve or synthetic antibody libraries by phage display. In this study, library techniques and phage display selection were applied in order to optimise binding scaffolds and antigen recognition of antibodies, and to evolve new and improved bioaffinity reagents. Antibody libraries were generated by random and targeted mutagenesis. Expression and stability were mainly optimised by the random methods whereas targeted randomisation of the binding site residues was used for optimising the binding properties. Trinucleotide mutagenesis allowed design of defined randomisation patterns for a synthetic antibody library. Improved clones were selected by phage display. Capture by a specific anti- DHPS antibody was exploited in the selection of improved phage display of DHPS. Efficient selection for stability was established by combining phage display selection with denaturation under reducing conditions. Broad-specific binding of a generic anti-sulfonamide antibody was improved by selection with one of the weakest binding sulfonamides. In addition, p9 based phage display was studied in affinity selection from the synthetic library. A TIM barrel protein DHPS was engineered for efficient phage display by combining cysteinereplacement with random mutagenesis. The resulting clone allows use of phage display in further engineering of DHPS and possibly use as an alternative-binding scaffold. An anti-TSH scFv fragment, cloned from a monoclonal antibody, was engineered for improved stability to better suite an immunoassay. The improved scFv tolerates 8 – 9 °C higher temperature than the parental scFv and should have sufficient stability to be used in an immunoanalyser with incubation at 36 °C. The anti-TSH scFv fragment was compared with the corresponding Fab fragment and the parental monoclonal antibody as a capturing reagent in a rapid 5-min immunoassay for TSH. The scFv fragment provided some benefits over the conventionally used Mab in anayte-binding capacity and assay kinetics. However, the recombinant Fab fragment, which had similar kinetics to the scFv, provided a more sensitive and reliable assay than the scFv. Another cloned scFv fragment was engineered in order to improve broad-specific recognition of sulfonamides. The improved antibody detects different sulfonamides at concentrations below the maximum residue limit (100 μg/kg in EU and USA) and allows simultaneous screening of different sulfonamide drug residues. Finally, a synthetic antibody library was constructed and new antibodies were generated and affinity matured entirely in vitro. These results illuminate the possibilities of phage display and antibody engineering for generation and optimisation of binding reagents in vitro and indicate the potential of recombinant antibodies as affinity reagents in immunoassays.

Jätevesien puhdistus jätevesialtaissa jäädyttämällä - Kartoitus potentiaalisista sovellutuksista Suomessa

Uusia jäteveden puhdistusprosesseja kartoitetaan Suomessakin esimerkiksi kiristyvien päästömääräyksien vuoksi sekä parempia kustannus- ja energiatehokkuuksia tavoiteltaessa. Jätevesien puhdistus on Suomessa jo nykyään hyvällä tasolla, mutta muun muassa raskasmetallien, torjunta-aineiden, hormonien ja lääkeaineiden pitoisuuksien kasvut jätevesissä asettavat haasteita nykyisin käytössä oleville jäteveden puhdistusmenetelmille, sillä niitä ei ole suunniteltu näiden aineiden talteenottoon ja suurin osa aineista jää veteen. Lisäksi jätevedet halutaan nähdä enemmänkin resurssina kuin jätteenä, josta voidaan ottaa talteen hyödyllisiä komponentteja, kuten suoloja. Alueilla, joissa vuorokauden keskilämpötila pysyttelee edes osan vuodesta pakkasella, veden luonnollista jäätymisprosessia voidaan käyttää hyväksi jäteveden puhdistuksessa. Tässä työssä selvitettiin kiteytymisen teorian ja aikaisempien tutkimusten avulla, millaisten jätevesien puhdistukseen jäädytyskiteytys sopii sekä pohdittiin menetelmän potentiaalisia sovelluskohteita Suomessa. Jäädytyskiteytyksen todettiin olevan turvallinen ja energiatehokas ratkaisu monien koostumukseltaan erilaisien jätevesien puhdistukseen. Menetelmällä voitaneen puhdistaa öljyisiä, orgaanisia ja/tai epäorgaanisia epäpuhtauksia tai raskasmetalleja sisältäviä sekä myrkyllisiä jätevesiä. Olosuhteet prosessille ovat parhaat Pohjois-Suomessa, jossa vuorokauden keskilämpötila pysyttelee nollan alapuolella noin seitsemän kuukautta vuodesta. Etelä-Suomessa vastaava luku on kolme. Menetelmän potentiaalisia sovelluskohteita ovat esimerkiksi kaivosteollisuuden ja kaatopaikkojen jätevedet, joiden puhdistukseen jäädytyskiteytys saattaisi soveltua erinomaisesti. Jäädyttämällä voitaisiin myös puhdistaa tekstiili- ja nahkateollisuuden jätevesiä, sillä niiden sisältämien väriaineiden erottaminen vedestä on perinteisillä jäteveden puhdistusmenetelmillä usein vaikeaa tai jopa mahdotonta. Suolojen kiteyttämiseen vaadittavia korkeampia suolapitoisuuksia todettiin löytyvän lähinnä membraaniprosessien, kuten käänteisosmoosin, rejektivesistä. Sopivimmat eutektiset olosuhteet kiteyttämiseen ovat natriumsulfaatilla, kaliumsulfaatilla ja natriumkarbonaatilla. Veden luonnollisen jäätymisprosessin hyödyntäminen jätevedenpuhdistuksessa on huomionarvoinen idea. Prosessin käyttöönottoa haittaavat esimerkiksi korkeat investointikustannukset, mutta ne tulevat todennäköisesti ajan myötä teknologian kehittyessä laskemaan. Lisäksi monet prosessiin liittyvät käytännön asiat ovat vielä tutkimuksen alla. On myös huomattava, että Suomessakaan lämpötila ei pysyttele koko vuotta pakkasella, joten jäädytyksen rinnalla on oltava jokin toinen prosessi, jolla jätevedet puhdistetaan lämpötilan ollessa nollan yläpuolella.

VAP-1 as drug target in inflammation : structural features determining ligand interactions

Vascular adhesion protein-1 (VAP-1), which belongs to the copper amine oxidases (CAOs), is a validated drug target in inflammatory diseases. Inhibition of VAP-1 blocks the leukocyte trafficking to sites of inflammation and alleviates inflammatory reactions. In this study, a novel set of potent pyridazinone inhibitors is presented together with their X-ray structure complexes with VAP-1. The crystal structure of serum VAP-1 (sVAP-1) revealed an imidazole binding site in the active site channel and, analogously, the pyridazinone inhibitors were designed to bind into the channel. This is the first time human VAP-1 has been crystallized with a reversible inhibitor and the structures reveal detailed information of the binding mode on the atomic level. Similarly to some earlier studied inhibitors of human VAP-1, the designed pyridazinone inhibitors bind rodent VAP-1 with a lower affinity than human VAP-1. Therefore, we made homology models of rodent VAP-1 and compared human and rodent enzymes to determine differences that might affect the inhibitor binding. The comparison of the crystal structures of the human VAP-1 and the mouse VAP-1 homology model revealed key differences important for the species specific binding properties. In general, the channel in mouse VAP-1 is more narrow and polar than the channel in human VAP-1, which is wider and more hydrophobic. The differences are located in the channel leading to the active site, as well as, in the entrance to the active site channel. The information obtained from these studies is of great importance for the development and design of drugs blocking the activity of human VAP-1, as rodents are often used for in vivo testing of candidate drugs. In order to gain more insight into the selective binding properties of the different CAOs in one species a comprehensive evolutionary study of mammalian CAOs was performed. We found that CAOs can be classified into sub-families according to the residues X1 and X2 of the Thr/Ser-X1-X2-Asn-Tyr-Asp active site motif. In the phylogenetic tree, CAOs group into diamine oxidase, retina specific amine oxidase and VAP-1/serum amine oxidase clades based on the residue in the position X2. We also found that VAP-1 and SAO can be further differentiated based on the residue in the position X1. This is the first large-scale comparison of CAO sequences, which explains some of the reasons for the unique substrate specificities within the CAO family.

Probing MST1 Kinase Sequence and Structure for Rational Drug Discovery (Targeting diabetes by blocking protein-protein interactions)

Apoptotic beta cell death is an underlying cause majorly for type I and to a lesser extent for type II diabetes. Recently, MST1 kinase was identified as a key apoptotic agent in diabetic condition. In this study, I have examined MST1 and closely related kinases namely, MST2, MST3 and MST4, aiming to tackle diabetes by exploring ways to selectively block MST1 kinase activity. The first investigation was directed towards evaluating possibilities of selectively blocking the ATP binding site of MST1 kinase that is essential for the activity of the enzymes. Structure and sequence analyses of this site however revealed a near absolute conservation between the MSTs and very few changes with other kinases. The observed residue variations also displayed similar physicochemical properties making it hard for selective inhibition of the enzyme. Second, possibilities for allosteric inhibition of the enzyme were evaluated. Analysis of the recognized allosteric site also posed the same problem as the MSTs shared almost all of the same residues. The third analysis was made on the SARAH domain, which is required for the dimerization and activation of MST1 and MST2 kinases. MST3 and MST4 lack this domain, hence selectivity against these two kinases can be achieved. Other proteins with SARAH domains such as the RASSF proteins were also examined. Their interaction with the MST1 SARAH domain were evaluated to mimic their binding pattern and design a peptide inhibitor that interferes with MST1 SARAH dimerization. In molecular simulations the RASSF5 SARAH domain was shown to strongly interact with the MST1 SARAH domain and possibly preventing MST1 SARAH dimerization. Based on this, the peptidic inhibitor was suggested to be based on the sequence of RASSF5 SARAH domain. Since the MST2 kinase also interacts with RASSF5 SARAH domain, absolute selectivity might not be achieved.