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.

Biomarkkerit sepsiksen diagnostiikassa sekä Kvantitatiivisen prokalsitoniini-immunomäärityksen pystyttäminen ja optimointi point-of-care-ympäristöön

Termiä sepsis käytetään infektion aiheuttamasta yleisestä tulehdusreaktio-oireyhtymästä. Biokemiallinen merkkiaine eli biomarkkeri on aine, joka objektiivisesti mitattuna ja arvioituna toimii indikaattorina erottaen toisistaan normaalit biologiset prosessit ja patogeeniset prosessit. Lisäksi biomarkkeri saattaa osoittaa farmakologisen vasteen lääkehoidolle. Tutkielman kirjallisessa osassa on esitelty ja verrattu toisiinsa jo kliinisessä käytössä olevia sekä lupaavia, vielä tutkimusvaiheessa olevia, sepsiksen biomerkkiaineita. Lisäksi tutkielmassa on tarkasteltu sepsiksen todentamisen, tunnistamisen ja hoidon tulevaisuudennäkymiä. Prokalsitoniini (PCT) on eräs jo kliinisessä käytössä oleva sepsiksen biomerkkiaine. Tutkielman kokeellisessa osassa pystytettiin teho-osastoille suunnattu pikatesti PCT:n mittaamiseen aikaerotteista fluoresenssiteknologiaa hyödyntävälle AQT-järjestelmälle (DHR Finland Oy, Innotrac Diagnostics, Suomi). Järjestelmä mittaa 20–30 minuutissa biomerkkiainepitoisuuden. Tavoitteena oli löytää sopiva vasta-ainepari ja pystyttää kaksipuoleinen immunomääritys, jonka analyyttinen herkkyys on alle 0,02 ng/ml:ssa. Työssä tutkittiin biotinyloidun kokonaisen vasta-aineen ja paikkaspesifisesti biotinyloidun antigeenia sitovan fragmentin (engl. fragment antigen binding, Fab) toimivuutta sitojamolekyylinä. Lisäksi työssä verrattiin kahden eri leimamolekyylin, 9 hampaisen europiumkelaatin sekä Innotracissa kehitetyn tähtikelaatin, toimivuutta PCT-immunomäärityksessä. Vasta-aineiden mahdolliset ristireaktiot testattiin ja määritystä testattiin alustavasti eri näytematriiseissa. Työssä löydettiin 5 vasta-aineparia, joiden analyyttinen herkkyys oli alle 0,02 ng/ml:ssa. Aiemmin testattuja vasta-ainepareja parempi analyyttinen herkkyys johtui todennäköisesti yhden uuden leimavasta-aineen paremmasta affiniteetistä. Tähtikelaatilla leimatulla vasta-aineella analyyttinen herkkyys parani lähes viisinkertaisesti, kun sitä verrattiin 9-hampaisella euroopiumkelaatilla leimatun vasta-aineen määritykseen. Havaittiin myös, että Fab-fragmentilla mitattiin määrityksissä lähes kaksi kertaa suuremmat signaali-taustasuhteet, kun sitä verrattiin kokonaiseen vasta-aineeseen, tietyllä vasta-aineparilla. Tämä johtui luultavasti steeristen esteiden vähenemisestä. Näytematriisilla ei todettu olevan vaikutusta määritykseen, eikä ristireaktioita havaittu. Kehitetyn määrityksen suoriutumista verrattiin markkinoilla olevaan BRAHMS AG:n (Saksa) PCT-määritykseen käyttäen potilasnäytteitä. Korrelaatiokertoimet potilasnäytevertailuissa olivat 0,965–0,989 ja näytteiden mitatuissa pitoisuuksissa oli tasoero.

Streptavidin - A Versatile Binding Protein for Solid-Phase Immunoassays

Streptavidin, a tetrameric protein secreted by Streptomyces avidinii, binds tightly to a small growth factor biotin. One of the numerous applications of this high-affinity system comprises the streptavidin-coated surfaces of bioanalytical assays which serve as universal binders for straightforward immobilization of any biotinylated molecule. Proteins can be immobilized with a lower risk of denaturation using streptavidin-biotin technology in contrast to direct passive adsorption. The purpose of this study was to characterize the properties and effects of streptavidin-coated binding surfaces on the performance of solid-phase immunoassays and to investigate the contributions of surface modifications. Various characterization tools and methods established in the study enabled the convenient monitoring and binding capacity determination of streptavidin-coated surfaces. The schematic modeling of the monolayer surface and the quantification of adsorbed streptavidin disclosed the possibilities and the limits of passive adsorption. The defined yield of 250 ng/cm² represented approximately 65 % coverage compared with a modelled complete monolayer, which is consistent with theoretical surface models. Modifications such as polymerization and chemical activation of streptavidin resulted in a close to 10-fold increase in the biotin-binding densities of the surface compared with the regular streptavidin coating. In addition, the stability of the surface against leaching was improved by chemical modification. The increased binding densities and capacities enabled wider high-end dynamic ranges in the solid-phase immunoassays, especially when using the fragments of the capture antibodies instead of intact antibodies for the binding of the antigen. The binding capacity of the streptavidin surface was not, by definition, predictive of the low-end performance of the immunoassays nor the assay sensitivity. Other features such as non-specific binding, variation and leaching turned out to be more relevant. The immunoassays that use a direct surface readout measurement of time-resolved fluorescence from a washed surface are dependent on the density of the labeled antibodies in a defined area on the surface. The binding surface was condensed into a spot by coating streptavidin in liquid droplets into special microtiter wells holding a small circular indentation at the bottom. The condensed binding area enabled a denser packing of the labeled antibodies on the surface. This resulted in a 5 - 6-fold increase in the signal-to-background ratios and an equivalent improvement in the detection limits of the solid-phase immunoassays. This work proved that the properties of the streptavidin-coated surfaces can be modified and that the defined properties of the streptavidin-based immunocapture surfaces contribute to the performance of heterogeneous immunoassays.

Collagen binding integrins and cancer testis antigens in prostate cancer and melanoma

Camilla Pelo Collagen Binding Integrins and Cancer Testis Antigens in Prostate Cancer and Melanoma Department of Biochemistry, MediCity Research Laboratory, University of Turku, Finland Annales Universitatis Turkuensis, Painosalama Oy, Turku, Finland 2016 ABSTRACT Prostate cancer is the second most common cancer in men worldwide. The incidence of melanoma, in turn, is increasing faster than any other cancer incidences. In Finland, more than 5000 prostate cancer and 1200 new melanoma cases are diagnosed each year. One approach to further understand the cellular processes involved in prostate cancer and melanoma is to gain better knowledge about alterations in gene expression and their potential impact on the progression of the diseases. This thesis is focused on expression studies in two gene families; integrins and cancer testis antigens (CT antigens), in human prostate adenocarcinoma and advanced human melanoma. Integrins are heterodimeric transmembrane receptors which regulate many important cellular processes such as cell proliferation, migration and survival. CT antigens are frequently expressed in different types of cancers, but are only expressed in testis in healthy individuals. CT antigens are also highly immunogenic proteins. Due to the properties mentioned above, integrins and CT antigens can function as target molecules for the development of cancer diagnostics and drugs. One of the main purposes of this thesis was to study the expression of the four collagen binding integrins α1β1, α2β1, α10β1, α11β1 and the cancer testis antigen 16 (CT16) in cancer cell lines and human tissues of prostate cancer and metastatic melanoma. Additional aims included studies on the biological role of CT16 and the abundance of CT16 in sera of advanced melanoma patients. The prognostic and diagnostic significance of CT16 and the collagen binding integrins were also evaluated. Expression studies on collagen binding integrins and the CT antigen CT16 in melanoma and prostate cancer were limited and the biological role of CT16 was unknown. In this thesis, the expression levels of α2β1 and α11β1 were found to be significantly altered in prostate cancer tissues. Integrin α2β1 decreased gradually during disease progression while α11 was elevated in prostate carcinoma compared to healthy tissues. In advanced melanoma, enhanced levels of α2 were associated with a significant shorter overall survival in advanced melanoma. In this thesis, CT16 was identified as a frequently expressed melanoma CT antigen with an anti-apoptotic function. To conclude, this thesis presents α2β1 and CT16, as potential and promising biomarkers for advanced melanoma. This thesis reports also the first functional study of CT16. Keywords: Collagen binding integrins, α1β1, α2β1, α10β1, α11β1, Cancer Testis antigens, CT16, melanoma, prostate cancer, expression