20.500.12556/RUL-131705 Biokemijska in biofizikalna karakterizacija aktinoporinom podobnega porotvornega proteina iz morskega polža Conus andremenezi. Biochemical and biophysical characterisation of an actinoporin-like pore forming protein from the sea snail Conus andremenezi. Aktinoporini (AP) so α-porotvorni proteini, prisotni v strupu morskih ožigalkarjev. Ob stiku s tarčno membrano topne monomerne oblike teh proteinov oligomerizirajo in po konformacijski spremembi protomerov predrejo membrano, da nastane transmembranska pora. Tipični predstavniki AP se specifično vežejo na sfingomielin, prisoten v celičnih membranah, in ne vsebujejo cisteinskih aminokislinskih ostankov. Zaradi svojih lastnosti so vse bolj zanimivi za mnoge aplikacije, predvsem s področja biotehnologije in medicine. Ker je pri razvoju aplikacij ključnega pomena širok nabor porotvornih proteinov z različnimi lastnostmi, je poleg genskega ali proteinskega inženiringa izjemnega pomena tudi odkrivanje in določanje lastnosti novih AP in aktinoporinom podobnih proteinov (ALP). Protein CAND iz morskega polža Conus andremenezi, ki je bil v sklopu naloge prvič rekombinantno izražen in eksperimentalno okarakteriziran, je eden od predstavnikov ALP. V njegovi primarni zgradbi so prisotna značilna aminokislinska zaporedja AP. Napoved elementov sekundarne strukture prav tako kaže na visoko podobnost z AP. Za CAND smo z merjenjem spektra cirkularnega dikroizma določili temperaturo taljenja približno 41 °C, nanjo pa pozitivno vpliva višja koncentracija soli in nižje pH vrednosti pufra. pH ima tudi močan vpliv na hemolitično aktivnost proteina CAND, ki pa je pri nižjem pH nižja, v splošnem pa je aktivnost CAND nekaj velikostnih razredov nižja od aktivnosti FraC. Kot nekateri predstavniki ALP, ki so bili do sedaj že eksperimentalno okarakterizirani, tudi CAND ne kaže specifičnosti za sfingomielin, saj se veže tudi na druge lipide v membranah. Porotvorno aktivnost proteina CAND smo tudi pokazali s posnetkom pore proteina CAND na velikih unilamelarnih veziklih (LUV) z uporabo krio-elektronske mikroskopije. Izolacijo pore iz veziklov pa smo izvedli z raztapljanjem LUV z dodatkom detergenta DDM in ločevanjem na velikostno izključitveni kromatografski koloni. Actinoporins (AP) are a group of α-pore-forming proteins found in the venom of sea anemones. In contact with target membranes, monomeric proteins oligomerize to form transmembrane pores through conformational change of the N-terminal α-helices. Typically, AP are known for their lack of cysteine residues and for binding to sphingomyelin, found in cell membranes. Their distinctive characteristics make them promising candidates for applications in biotechnology and medicine. Protein and genetic engineering have an important role in development of such applications, however there is also a fundamental demand for discovery and characterization of new AP and actinoporin-like proteins (ALP). In the scope of this thesis, we cloned a gene encoding protein CAND, ALP from Conus andremenezi, and recombinantly expressed it in Escherichia coli, purified and experimentally characterized for the first time. Amino acid sequence of CAND, as well as structural elements of secondary structure show numerous similarities to AP. CAND has melting temperature (Tm), around 41 °C (measured with CD spectroscopy), however salt concentration and pH have a significant effect on it. Increasing the salt concentration and lowering the pH have a positive effect on Tm. pH also showed significant effect on haemolytical activity of CAND, which decreases with lower pH values, and is in general few orders of magnitude lower compared to FraC. Like some of the ALP representatives that have been experimentally characterized to date, CAND does not show specificity for sphingomyelin, as it also binds to other lipids in the membrane. The pore forming activity of CAND protein was demonstrated by recording the pore of the CAND protein on large unilamilary vesicles (LUV) using cryo-electron microscopy. Pore isolation was performed by disruption of LUV with detergent DDM and subsequent separation with size exclusion chromatography. biotehnologija porotvorni protein aktinoporinom podobni protein biotechnology pore-forming protein actinoporin-like protein true false false [L. Fajdiga] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2021-10-01 10:21:34 2021-10-01 10:21:39 2022-09-10 04:13:16 0000-00-00 00:00:00 2021 0 Ljubljana 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577.112:594.3(043.2) 199325 78990083 20767.pdf 20767.pdf 1 89DE6A55B3A6D44C8EB73143492FA97E e2056c08e92833de14128f819b1d0eb1216350840e6d36f8b37ea146f330f2c0 50f7ba0c-2290-11ec-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=149095 Biotehniška fakulteta 0 0 0