Optimization of Escherichia coli expression, purification and characterization of antibody fragments

master's thesis

• Title (croatian): Optimizacija ekspresije, pročišćavanja i karakterizacije fragmenata protutijela iz bakterije Escherichia coli
• Author: Mirna Nakić
• Mentor: Igor Stuparević (mentor)
• Committee member: Igor Stuparević (predsjednik povjerenstva)
• Committee member: Višnja Besendorfer (član povjerenstva)
• Committee member: Ivana Ivančić Baće (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2023-07-03, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Camelid VHHs, commonly known as nanobodies, have gained significant attention among researchers due to their distinct advantages over traditional antibodies. These advantages include their small size and the presence of only one disulfide bond as the sole post-translational modification, which makes them highly amenable to production in microbial systems. However, producing disulfide bonded proteins in a reducing cytoplasm of Escherichia coli is a challenging endeavor. So, several techniques were employed with the goal of optimizing the production of VHH, such as fusion with a redox active and/or chaperone protein as well as utilizing a redox engineered (Origami™) strain that possesses a more oxidative cytoplasm. The highest yield of soluble VHH fusion protein was obtained with thioredoxin (Trx) as the fusion partner in BL21 (DE3) strain of E. coli. This result is indicative of the robust chaperone activity of thioredoxin which assists the VHH to reach its native conformation. Fusion tag removal was performed by cleavage with tobacco etch virus (TEV) protease which targets a highly specific sequence within the fusion protein and once cleaved VHH was successfully obtained, characterization tests could proceed. It was revealed that the produced VHH does not contain the expected disulfide bridge which could potentially explain some of the observed properties of the VHH, such as inefficient renaturation and dimer formation.

Abstract (croatian)

Kamelidna nanotijela privukla su značajnu pozornost među istraživačima zbog svojih prednosti u odnosu na tradicionalna antitijela. Ove prednosti uključuju njihovu malu veličinu i prisutnost samo jedne disulfidne veze kao jedine post-translacijske modifikacije, što ih čini vrlo podložnim proizvodnji u mikrobnim sustavima. Međutim, proizvodnja proteina povezanih disulfidnom vezom u reducirajućoj citoplazmi Escherichiae coli izazovan je pothvat. Dakle, korišteno je nekoliko tehnika s ciljem optimiziranja proizvodnje nanotijela, kao što je fuzija s redoks aktivnim i/ili proteinom šaperonom kao i korištenje Origami™ soja za kojeg je redoks inženjerstvom postignuto da posjeduje citoplazmu oksidativnih svojstava. Najveći prinos topljivog fuzijskog proteina dobiven je s tioredoksinom (Trx) kao fuzijskim partnerom u BL21 (DE3) soju E. coli. Ovaj rezultat je pokazatelj snažne šaperonske aktivnosti tioredoksina koji pomaže nanotijelu da postigne svoju nativnu konformaciju. Uklanjanje fuzijske oznake provedeno je cijepanjem TEV (engl. tobacco etch virus) proteazom koja cilja visoko specifičnu sekvencu unutar fuzijskog proteina i nakon što je pocijepano nanotijelo uspješno dobiveno, testovi karakterizacije mogli su započeti. Otkriveno je da proizvedeno nanotijelo ne sadrži očekivani disulfidni most, što je rezultat koji bi potencijalno mogao objasniti neka od opaženih svojstava nanotijela, kao što su neučinkovita renaturacija i stvaranje dimera.

• Keywords: nanobody; soluble; disulfide bond; Escherichia coli
• Keywords (croatian): nanotijelo; topljiv; disulfidna veza; Escherichia coli
• Language: english
• URN:NBN: urn:nbn:hr:159:191157
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-07-03 09:46:05