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Generation and Evaluation of a Novel Affibody Library
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
2018 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Affinity proteins have found significant use as biopharmaceuticals. Development of scaffold proteins, such as the affibody molecule, have simplified engineering and production of high affinity binders. In this thesis, a new affibody library was designed and created with selected diversity for higher functionality. Trinucleotide synthesis and solid phase cloning was utilized for accurate diversity and assembly. The diversity was designed to mimic the binding domains of antibodies which are natural high affinity binders. The library reached a size of ten billion clones and was analyzed by deep sequencing and an array of bioinformatic tools. The library wasconfirmed to have high functionality and the actual diversity was shown to correlate well with theexpected diversity. Trinucleotide synthesis was shown to be more error prone than traditionalsynthesis, with single base errors shown to be the most common error with incorporation or exclusion of complete codons second. A new, smaller E. coli display vector was constructed for more effective cloning of affibody libraries. Selections from the library were carried out by both phage display and E. coli display towards HER2 and the two immune checkpoint proteins OX40 and PD-L1. Phage display was successfully used to reduce the complexity of the library.

Place, publisher, year, edition, pages
2018.
National Category
Natural Sciences Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-235501OAI: oai:DiVA.org:kth-235501DiVA, id: diva2:1251417
Available from: 2018-09-27 Created: 2018-09-27 Last updated: 2018-09-27Bibliographically approved

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