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Reducerad aggregering av affibodymolekyler
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems.
2022 (Swedish)Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesisAlternative title
Reducing Aggregation of Affibody Molecules (English)
Abstract [sv]

I dagsläget utvecklas ständigt nya läkemedel med hjälp av affinitetsprotein. På 90-talet skapades ett nytt sådant protein utvunnet från Protein A på KTH, den så kallade affibodyn. Att arbeta med affibodies är ofta fördelaktigt eftersom de är lätta producera samtidigt som de delar många av de väsentliga egenskaperna hos en antikropp. Affibodies är även kända för att vara mycket stabila, vilket betyder att de i de flesta fall klarar av att vecka sig tillbaka till korrekt struktur efter denaturering. Tyvärr är det inte alltid fallet och ett exempel på det är de affibodies i biblioteket som konstruerats för att binda till spike 1 på SARS-CoV-2 i Johan Rockbergs labb på Proteinteknologi i AlbaNova. I detta projekt har affibodyn ZS1-ORANGE-22, från biblioteket, producerats i tre nya expressionsvektorer som tidigare inte prövats. Vektorerna innehåller olika element som tidigare påvisat öka lösligheten av protein. Resultatet från produktionen visar att vektorn som tidigare använts för produktion av affibodyn producerade den största mängden protein. Däremot uppvisade de tre nya vektorerna som utvärderats i projektet ett större procentuellt utbyte protein efter rening. I kombination med detta har även en studie kring hur buffertars komposition inverkar på proteinlösligheten gjorts. Resultat från denna utvärdering visar att tillsatsen av L-arginin och L-Glutaminsyra till PBS ökar lösligheten av proteinet, men att det även kan en inverkan på dess struktur som gör det mindre lämpligt för tillämpningar samt analys.

Abstract [en]

Today new drugs based on affinity proteins are constantly developed. In the 90s a new type of affinity protein was discovered from Protein A at KTH, the affibody. Affibodies are often favorable to work with since they are easy to produce and share a lot of essential characteristics with antibodies. They are also known for being stable, meaning they can refold correctly after being denatured. However, this is not always the case and an example of this is the library of affibodies designed to bind to spike 1 on SARS-CoV-2 in Johan Rockbergs lab at Proteintechnology in AlbaNova. In this project the affibody ZS1-ORANGE-22, from said library, has been produced in three new expression vectors which all have elements that previously have shown to help with precipitation. The results show that the vector that has previously been used to produce the affibody produces a larger quantity of protein. However the three new vectors that have been evaluated in this project give a larger percentage yield after purification. In combination with this, a study on how buffer compositions impact protein solubility has been conducted. Results have shown that adding L-Arginine and L-Glutamic acid to a PBS buffer has positively impacted the solubility of the proteins. However, this may also affect their structure making them less suitable for application and analysis. 

Place, publisher, year, edition, pages
2022. , p. 54
Series
TRITA-CBH-GRU ; 2022:145
Keywords [en]
Affibody, solubility, precipitation, aggregation prone, concentration determination
Keywords [sv]
Affibody, löslighet, aggregerande protein, koncentrationsbestämning
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-316347OAI: oai:DiVA.org:kth-316347DiVA, id: diva2:1687457
Subject / course
Medical Engineering
Educational program
Master of Science in Engineering - Medical Engineering
Supervisors
Examiners
Available from: 2022-08-16 Created: 2022-08-15 Last updated: 2022-08-16Bibliographically approved

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