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Aktinoporini in aktinoporinom podobni proteini predstavljajo veliko skupino α-porotvornih proteinov. Ob vezavi na membrane pride do konformacijske spremembe monomerne oblike, sledi oligomerizacija, kjer pride do povezovanja večjega števila monomerov in tvorbe pore. Velik napredek je narejen na področju uporabe por kot biosenzorjev, kjer so zahteve po robustnih porah, z možnostjo nadaljnjih modifikacij za spreminjanje njihovih lastnosti. V okviru magistrske naloge smo v celicah E.coli BL21 (DE3), izrazili 10 konstruktov aktinoporinskega homologa osnovanega na EST zaporedju iz Orbicella faveolata (Fav). Proteine smo izolirali in očistili z nikelj-afinitetno kromatografijo in njihovo porotvornost potrdili z izvedbo testa hemolize. Sposobnost oligomerizacije in vstavljanja v lipidne membrane smo potrdili tudi na sistemu Orbit mini, kjer smo v membrano DOPC:SM uspešno vstavili vse konstrukte in tako uspešno izvedli biofizikalno karakterizacijo. Za izbor konstrukta, ki izkazuje najmanjši šum električnega toka v primerjavi s kontrolnim konstruktom smo izvedli predhodno tvorbo por in analizo s sistemom Orbit mini na stabilnejših membranah iz lipidov DPhPC. Konstrukt z najmanjšim šumom smo uporabili za poskuse zaznavanja. Za uspešno zaznavanje je bilo potrebno optimizirati električno napetost, njeno polariteto, puferske raztopine in koncentracijo proteina, da smo kljub visoki tendenci do vstavljanja v membrane, pridobili meritve z nizkim tokom, ki odraža prisotnost ene ali dveh por. Ob dodatku histonov H3.1 in H4 so bile vidne specifične blokade, ki omogočajo ločbo med dvema histonskima variantama, ki se v glavnem razlikujeta le v neto naboju in dolžini N-končnega pozitivno nabitega histonskega repka. Actinoporins and actinoporin-like proteins represent a large group of α-pore-forming proteins. Upon binding to membranes, a conformational change of the monomer form occurs, followed by oligomerization, where a larger number of monomers assemble to form a pore. Lots of research and development is being conducted to be able to use pores as biosensors. Such application requires a robust pore, which can be further modified to augment its properties. As part of the Master's thesis, we expressed 10 actinoporin homolog constructs based on the EST sequence from Orbicella faveolata (Fav) in E.coli BL21 (DE3) cells. Proteins were isolated and purified by nickel-affinity chromatography, and their pore-forming ability was confirmed by performing a hemolysis test. The ability to oligomerize and insert into lipid membranes was also confirmed on the Orbit mini system, where we successfully inserted all constructs into the DOPC:SM membrane. We were able to perform biophysical characterization. To select the construct that exhibits the smallest electric current noise compared to the control construct, we performed preliminary pore formation and analysis with the Orbit mini system on more stable membranes made of DPhPC lipids. The construct with the lowest noise was used for histone detection experiments. For successful detection, it was necessary to optimize the electric voltage, its polarity, buffer solutions and protein concentration, so that despite Fav’s high tendency to insert into the membranes, we obtained measurements with a low current, which reflects the presence of only one or two pores. Upon the addition of histones H3.1 and H4, different types of blockages were observed, which indicates the ability to separate between the two histone variants, which mainly differ only in the net charge and the length of the N-terminal positively charged histone tail. |
