Produkcija in karakterizacija modificiranih oblik proteinskih plinskih veziklov v E. coli

• Main Author: Šprager, Ernest
• Other Authors: Benčina, Mojca
• Format: Master Thesis
• Language: Slovenian
• Published: 2020
• Subjects: proteinski plinski vezikli; GvpC; mutageneza skupka gvp; ultrazvok; protein gas vesicles; gvp cluster mutagenesis; ultrasound; Domen; sami
• Online Access: https://repozitorij.uni-lj.si/IzpisGradiva.php?id=116808; https://repozitorij.uni-lj.si/Dokument.php?id=130496&dn=; https://plus.si.cobiss.net/opac7/bib/19613187?lang=sl

Proteinski plinski vezikli so cilindrične ali vretenaste votle nanostrukture prokariontskih mikroorganizmov s premerom 45 – 200 nm in dolžino 100 – 200 nm, napolnjene s plini in obdane s proteinsko steno, debelo 2 nm. Glavni strukturni protein predstavlja GvpA (ali paralogni GvpB), pri določenih vrstah mikroorganizmov pa plinski vezikli vsebujejo še pomožni strukturni protein GvpC, ki nekovalentno prečno poveže GvpA in proteinske plinske vezikle strukturno ojača. GvpC je možno rekombinatno izraziti z raznimi fuzijskimi partnerji in tako pripraviti funkcionalizirane plinske vezikle. V magistrskem delu je opisana priprava proteinskih plinskih veziklov z različnimi fuzijskimi oblikami GvpC v bakterijskih celicah Escherichia coli in karakterizacija njihove strukture ter oblike s presevno elektronsko mikroskopijo. Pokazali smo, da dodajanje proteinskih domen, kot je eGFP, h GvpC ne vpliva na njegovo sposobnost vezave na GvpB, hkrati pa se v tem primeru eGFP tudi pravilno zvije. S hkratnim heterolognim izražanjem skupka gvp iz Bacillus megaterium in modificiranih oblik GvpC iz Anabaena flos-aquae je možno v E. coli pripraviti poljubno funkcionalizirane proteinske plinske vezikle. Morfološko gledano so proteinski plinski vezikli iz B. megaterium pretežno vretenasti, medtem ko dodatek GvpC-RGD (RGD je peptidno zaporedje z visoko afiniteto do integrinov) povzroči nastanek daljših in cilindrično oblikovanih veziklov, ki so po velikosti in obliki bolj podobni nativnim iz A. flos-aquae. S fuzijo peptidnega zaporedja RGD in GvpC smo dosegli pripenjanje veziklov na membrano sesalskih celic HEK293, za samo upodabljanje vezave pa smo uporabili fluorescenčno mikroskopijo in fuzijski GvpC z eGFP in zaporedjem RGD. Z analizama SDS-PAGE in WB izoliranih in očiščenih proteinskih plinskih veziklov smo razkrili njihovo kompleksno proteinsko sestavo. Proteinski plinski vezikli imajo primerljive akustične lastnosti mikromehurčkom in so uporabni kot kontrastna sredstva v ultrazvočnem slikanju. Preverili smo, ali s peptidom RGD funkcionalizirani proteinski plinski vezikli lahko delujejo kot ojačevalci ultrazvočne stimulacije mehansko občutljivih ionskih kanalov. Z vezikli občutljivosti celic na ultrazvok nismo povečali, smo pa zaznali proženje sistema pred stimulacijo že ob sami vezavi veziklov. Preverili smo vpliv izbrisa posameznih genov v skupku gvp B. megaterium na heterologno izgradnjo proteinskih plinskih veziklov v E. coli. Posredno preko ocenjevanja sposobnosti plavanja transformiranih bakterij smo identificirali potencialno nepotrebne gene gvpR, gvpN, gvpT, gvpU, po izbrisu gvpR in gvpU pa smo zaznali tudi izgrajene vezikle. Protein gas vesicles are cylindrical or spindle-shaped hollow nanostructures in prokaryotic microorganisms with a diameter of 45 - 200 nm and a length of 100 - 200 nm, filled with gas and formed by a 2 nm thick protein wall. The major structural protein is GvpA (or its paralog GvpB), while some microorganism's gas vesicles also contain second structural protein GvpC, which attaches to the GvpA and stabilizes the wall. GvpC can be recombinantly expressed with various fusion partners to produce functionalized gas vesicles. In this work, we present the preparation of protein gas vesicles with different fusion forms of GvpC in Escherichia coli bacterial cells and characterization of their structure and shape by transmission electron microscopy. We have shown that the addition of protein domains like eGFP to GvpC does not affect its ability to bind to GvpB but also allows for the proper folding of eGFP. Thus, it is possible to prepare functionalized gas vesicles in E. coli with simultaneous heterologous expression of gvp cluster from Bacillus megaterium and modified GvpC from Anabaena flos-aquae. Morphologically, gas vesicles from B. megaterium are predominantly spindle-shaped, whereas the addition of GvpC-RGD (RGD is a peptide sequence with high affinity for integrins) results in the formation of longer and cylindrically formed vesicles, which are more similar in size and shape to the native ones from A. flos-aquae. A fusion of the RGD peptide with GvpC allowed attachment of vesicles to the membrane of mammalian HEK293 cells, while fusion of GvpC with eGFP and RGD sequences was used for confirmation of gas vesicles binding to the membrane by fluorescence microscopy imaging. SDS-PAGE and WB analysis of isolated and purified protein gas vesicles revealed their complex protein composition. Protein gas vesicles have comparable acoustic properties to microbubbles and are useful as contrast agents in ultrasonic imaging. We have checked whether gas vesicles functionalized with RGD peptides can act as enhancers in ultrasonic stimulation of mechanosensitive ion channels. The vesicles did not increase the sensitivity of the cells to ultrasound, but we detected the triggering of the system before stimulation at the very time of vesicle binding. We examined the effect of deletion of individual genes in the B. megaterium gvp cluster on the heterologous production of protein gas vesicles in E. coli. Indirectly through the assessment of the floating ability of transformed bacteria, we identified potentially unessential genes gvpR, gvpN, gvpT gvpU, and upon deletion of gvpR and gvpU, we were able to detect functional gas vesicles.