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Priprava sintetičnih enodomenskih protiteles in njihovo označevanje z encimom sortaza A
ID Hribar, Urban (Author), ID Turk, Boris (Mentor) More about this mentor... This link opens in a new window

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Abstract
Sintetična enodomenska protitelesa (sybody-ji) so majhna enodomenska protitelesa, ki temeljijo na strukturi variabilnih regij kameloidnih protiteles s samo težko verigo (nanotelo). Z in vitro selekcijo lahko ustvarimo sybody-je proti poljubnim proteinom, dobra potencialna tarča zanje je katepsin B (KatB). Ta je prisoten v visokih koncentracijah v mikrookolju večih različnih rakov, kar pomeni, da bi se sybody-ji proti KatB lahko uporabili za vizualizacijo tumorjev. Zaradi nizke molske mase bi molekularne sonde na osnovi sybody-jev imele hiter očistek in posledično nižji signal ozadja. Pri magistrskem delu smo sybody »Sy7« proti človeškemu in mišjemu KatB označili s fluorescenčnim barvilom Cyanine 5.5 (Cy5.5), ki emitira v bližnjem infrardečem spektru (NIR). To smo dosegli tako, da smo s pomočjo maleimidne reakcije najprej s Cy5.5 označili peptid CLPETGY. Označeni peptid Cy5.5-CLPETGY smo nato encimsko pripeli na N-konec modificiranega Sy7 s pomočjo rekombinantne sortaze A (SorA). Označen sybody Sy7GGCy5.5 smo izolirali in potrdili, da se še vedno veže na KatB. Pripravo večjih količin Sy7GGCy5.5, potrebnih za nadaljnje raziskave, z uporabljeno metodo na žalost omejuje nizek izkoristek in zamudnost nekaterih korakov, kar pomeni da je potrebna nadaljnja optimizacija postopka.

Language:Slovenian
Keywords:Sybody, molekularna vizualizacija tumorskega mikrookolja, Cyanine 5.5, označevanje N-konca, sortaza A, katepsin B
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2022
PID:20.500.12556/RUL-142906 This link opens in a new window
COBISS.SI-ID:135290627 This link opens in a new window
Publication date in RUL:01.12.2022
Views:610
Downloads:60
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Secondary language

Language:English
Title:Preparation and enzymatic labeling of sybodies using sortase A
Abstract:
Sybodies (Synthetic single domain antibodies) are small single-domain antibodies, the structure of which is based on camelid heavy-chain-only antibody binding domains (Nanobodies). Sybodies that bind against specific proteins can be selected using only in vitro methods. High levels of Cathepsin B (KatB) are found in a wide variety of cancers, making it a promising target for the development of molecular probes for non-invasive imaging. Due to their small size sybody-based molecular probes would have rapid clearance and therefore low background signal. Here we labeled »Sy7«, a sybody targeting human and mouse KatB, with a near-infrared (NIR) fluorescent dye Cyanine 5.5 (Cy5.5). First, we labeled a CLPETGY peptide with Cy5.5 using a maleimide reaction. We then attached the labeled peptide Cy5.5CLPETGY to the N-terminus of a modified Sy7 sybody by sortase A-mediated reaction. We found that the labeled and purified Sy7GGCy5.5 still retains the ability to bind to KatB. Alas using the methods we employed, the production of greater amounts of Sy7GGCy5.5 is limited due to the low yield and long duration of some steps. Our methods, therefore, require further optimization.

Keywords:Sybody, molecular imaging of tumor microenvironment, Cyanine 5.5, N- terminal labeling, sortase A, cathepsin B

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