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Interactions of the Human Recombinant Proteins JUNO and IZUMO1

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Interactions of the Human Recombinant Proteins JUNO and IZUMO1 Interactions of the Human Recombinant Proteins JUNO and IZUMO1 A master thesis project by Emma Lundell Department of Industrial Biotechnology in cooperation with Spermosens AB Prof. Per Berglund Lic. Kushagr Punyani August – December 2019 Acknowledgements Jag vill tacka Per Berglund som har varit en fantastisk handledare och alltid varit ett bollplank när jag har behövt det. Thank you, Kush Punyani and Mohamad Takwa, for giving birth to the idea of an exciting project that came to be my master thesis. Tack Johan Nilvebrant och Per-Åke Nygren för all er hjälp med Biacore, era idéer och för allt ni lärt mig. Tack Adam för att du har korrekturläst och stöttat mig under de sista intensiva veckorna. 2 Sammanfattning Det uppskattas att 15% av alla par världen över lider av infertilitet. Ungefär hälften beror på manlig infertilitet och 40% av dessa fall kan ännu inte förklaras. Därmed är nuvarande metoder för att diagnostisera manlig infertilitet otillräckliga och ytterligare tekniker behövs. En lyckad befruktning kräver att spermierna uttrycker membranproteinet Izumo1 som måste känna igen dess receptorprotein Juno, belägen vid ytan av äggmembranet. Bindningen mellan Juno och Izumo1 är essentiell för befruktning hos däggdjur då den bidrar till att gameterna binder och skapar en ny distinkt organism. Juno är ett relativt nyupptäckt protein och mekanismen med Izumo1 är fortfarande okänd. Ett nystartat företag vid namn Spermosens vill mäta interaktionen mellan Juno och Izumo1 i ett nytt diagnostikverktyg som ska diagnostisera manlig infertilitet. Tanken är att Juno ska immobiliseras på guld-nanopartiklar och användas för att mäta interaktionen med spermaprover. Det nya verktyget ska hjälpa par att fastställa felet i befruktningen, vilket som en följd skulle hjälpa paret att välja lämplig assisterad reproduktionsmetod. I utvecklingen av det nya diagnostikverktyget behöver det konfirmeras att den Juno som används i enheten kan binda korrekt till mänskligt Izumo1. Därför måste interaktionerna mellan de mänskliga, rekombinanta proteinerna Juno och Izumo1 mätas och karakteriseras. Syftet med detta projekt var att utveckla en metod för att immobilisera Juno på guld-nanopartiklar och sedan mäta interaktionerna med Izumo1 genom UV-Vis spektroskopi. Detta är teoretiskt möjligt eftersom guld-nanopartiklarna framkallar ett fenomen som kallas lokaliserad ytplasmonresonans som varierar beroende på storleken på guld-nanopartikelkomplexet. Immobiliseringsmetoden var en process som involverade flera steg som designades, polerades och förbättrades under arbetets gång. Dithiobis(C2-NTA) konjugerades till guldytan och koboltjoner konjugerades till NTA. Det sista steget som innebar konjugering av Juno till kobolt genom en His-tag lyckades inte, och interaktionerna kunde därför inte mätas genom denna metod. Istället mättes protein-protein-interaktionen genom SPR-mätningar med Biacore, ett instrument som också är baserat på ytplasmonresonans. Interaktioner mellan Izumo1 och Juno kunde uppmätas både vid användning av Juno producerad från E. coli och från däggdjursceller. Dissociationskonstanten (Kd) beräknades till 7-33 nM (för Juno och Izumo1 producerade i däggdjursceller) vilket kan jämföras med ett experiment från 2016 där 48 nM beräknades. Ett mer exakt Kd kunde inte fastställas och en trolig anledning till detta var att regenereringen av sensorytan som utfördes med NaOH varierade i effektivitet, vilket ledde till en osäkerhet då ytförhållandena kan ha varierat mellan mätningarna. De två Juno- proteinerna, som är producerade i olika organismer, visade två skilda affinitetsprofiler med Izumo1 vilket tyder på att glykosyleringen påverkar bindningsmekanismen mellan Juno och Izumo1. 3 Abstract It is estimated that 15% of all couples worldwide suffer from infertility. Roughly half is male-factor infertility and 40% of these cases cannot be explained. Thus, current methods for diagnosing male infertility are not enough and further techniques are needed. To have a successful fertilisation event, it is required that the sperm expresses membrane surface-protein Izumo1 which must recognise its counterpart protein Juno, located at the surface of the egg membrane. The recognition step between Juno and Izumo1 is essential in mammalian fertilisation for the gametes to bind and start the creation of a new distinct organism, but the molecular mechanism is still unknown. A start-up company named Spermosens want to measure the Juno-Izumo1 interaction in a new diagnostic device designed to diagnose male infertility. The idea is to have Juno immobilised on gold nanoparticles and measure the interaction between Juno and various semen samples. The new device is supposed to help couples pin-point the procreation issue which would help in the selection of suitable assisted reproductive technology. In the development of the new device, it had to be established that the Juno used in the device will bind correctly to human Izumo1. Therefore, the interactions between the human recombinant proteins Juno and Izumo1 had to be measured and characterized. The objectives of this project were to develop a method to immobilise Juno on gold nanoparticles and then measure the interactions with Izumo1 using UV-vis spectroscopy. This is theoretically possible since the gold nanoparticles exhibit a phenomenon called localized surface plasmon resonance that vary depending on the size of the gold nanoparticle-complex. The immobilisation procedure was a process involving several steps that were designed, polished and improved along the way. Dithiobis(C2-NTA) was conjugated to the gold surface and a cobalt ion was conjugated to the NTA. The last step involving conjugation of Juno to the cobalt through a His-tag was not succeeded, and the interactions could therefore not be measured this way. Instead, the protein-protein interaction was measured through SPR-measurements using Biacore, an instrument that is based on surface plasmon resonance as well. Interactions between Izumo1 and Juno could be detected using Juno produced in E. coli and in mammalian cells. The dissociation constant (Kd) could be calculated to 7-33 nM which can be compared to a previously published Kd of 48 nM. A more precise Kd could not be established, possibly due to that the regeneration of the sensor surface with NaOH varied in efficiency, leading to changing surface conditions during the measurements. The two Juno proteins, that were produced in different hosts, showed two different affinity profiles with Izumo1, which contributes to the suggestion that the glycosylation plays a role in the binding mechanism between Juno and Izumo1. 4 Table of Contents ABSTRACT ........................................................................................................................................................ 4 1 ABBREVIATIONS ....................................................................................................................................... 7 2 INTRODUCTION........................................................................................................................................ 8 3 BACKGROUND ......................................................................................................................................... 8 3.1 CURRENT DIAGNOSTIC METHODS ................................................................................................................... 8 3.2 FERTILISATION ............................................................................................................................................ 8 3.2.1 Juno ................................................................................................................................................... 9 3.2.2 Izumo1 ............................................................................................................................................. 10 3.3 ASSISTED REPRODUCTIVE TECHNOLOGY ......................................................................................................... 11 3.3.1 Traditional in vitro fertilisation ........................................................................................................ 11 3.3.2 Intracytoplasmic sperm injection .................................................................................................... 11 3.3.3 IVF success rates .............................................................................................................................. 11 3.3.4 Better ART-selection with new diagnostic device ............................................................................ 11 3.3.5 Kinetics & Affinity ............................................................................................................................ 13 3.4 OPTICAL PROPERTIES OF NOBLE METAL NANOPARTICLES ................................................................................... 14 3.5 SPR-BASED BIOSENSORS ............................................................................................................................ 16 3.5.1 Gold nanoparticles as biosensors .................................................................................................... 16 3.5.2 Biosensing with Biacore................................................................................................................... 17 4 OBJECTIVES ............................................................................................................................................ 19 5 METHODS & MATERIAL ........................................................................................................................
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