Uporaba peptidnih oznak za razlikovanje terapevtskih proteinov, predstavljenih na površini bakterije Lactococcus lactis

Rak debelega črevesa in danke (RDČD) predstavlja velik javnozdravstveni problem s pogosto slabo prognozo v primeru pozne diagnoze in terapije. Konvencionalne metode zdravljenja imajo številne slabosti in omejitve, kar spodbuja razvoj sodobnejših pristopov k zdravljenju, usmerjenih v ciljano dostavo učinkovin, kombinirano terapijo in teranostiko. Pomemben dejavnik tveganja za nastanek in napredovanje RDČD je vnetje v gastrointestinalnem traktu s posledično aktivacijo imunskih celic ter sproščanjem vnetnih citokinov. Z dostavo ustreznih protivnetnih učinkovin v črevesje lahko omilimo vnetje ter z njim povezane poškodbe sluznice in upočasnimo ali celo ustavimo progresijo RDČD, s hkratno dostavo vezalcev tumorsko specifičnih antigenov pa lahko dosežemo ciljano dostavo teh učinkovin. V sklopu raziskovalnega dela smo pripravili genske konstrukte in jih vstavili v plazmide pSDBA3b oziroma pNZDual za posamezno ali sočasno površinsko predstavitev dveh proteinov. V konstrukte smo vstavili gen za protein evazin (vezalec provnetnega IL-8) oziroma gen za protein afitin (vezalec tumorskega antigena EpCAM) ter zaporedje za peptidno oznako Cmyc ali Flag. Slednje so pomembno orodje v biokemiji, saj pripete na protein v kombinaciji s specifičnimi protitelesi omogočajo detekcijo, čiščenje in karakterizacijo rekombinantnih proteinov. Konstrukte smo izrazili v bakterijah L. lactis, ki so zaradi dolgoletne priznane varnosti in enostavne genske modifikacije primeren vektor za peroralno dostavo omenjenih terapevtskih proteinov. S pomočjo peptidnih oznak smo potrdili izražanje posameznih proteinov EVA-Cmyc, AFFI-Cmyc, EVA-Flag in AFFI-Flag oziroma njihovih kombinacij, dokazali njihovo prisotnost v celičnih lizatih in tudi na površini bakterij L. lactis, pri čemer je obseg površinske predstavitve variiral glede na vrsto proteina, uvedeno peptidno oznako in tudi glede na to, ali je bil protein izražen posamezno ali sočasno z drugim proteinom. Navsezadnje smo dokazali, da so bakterije s površinsko predstavljenimi evazini in afitini funkcionalne, torej sposobne vezave IL-8 in receptorja EpCAM. Zaradi splošnega neodobravanja GSO smo izražene proteine ločili od celic proizvajalk, jih nekovalentno pripeli na divje seve bakterij, ki niso bile gensko spremenjene in dokazali njihovo površinsko predstavitev. Celotno vrednotenje proteinov smo izvedli na osnovi peptidnih oznak in dokazali, da je njihova uporaba primeren, cenovno ugoden ter enostaven način za detekcijo in razlikovanje izraženih proteinov, kar je bil tudi osnovni namen magistrskega dela. Ugotovili smo, da oznake sicer sterično ovirajo vezavna mesta proteinov in vplivajo na njihovo funkcionalnost, a vendar prednosti njihove uporabe odtehtajo omenjene slabosti. Colorectal cancer (CRC) is a major public health problem with often poor prognosis in the case of a late diagnosis and therapy. Conventional methods for treatment have many disadvantages and limitations, which encourage the development of more modern approaches to treatment aimed at targeted active substance delivery, combination therapy and theranostics. An important risk factor for the development and progression of CRC is inflammation in the gastrointestinal tract with consequent activation of immune cells and the release of inflammatory cytokines. By delivering appropriate anti-inflammatory agents to the intestine, inflammation and associated mucosal damage can be alleviated and the progression of CRC can be slowed or even stopped. Concomitant delivery of tumor-specific antigen binders can achieve targeted delivery of these active ingredients. As part of the research work, we prepared gene constructs and inserted them into plasmids pSDBA3b or pNZDual for individual or simultaneous surface display of two proteins. The evasin protein gene (pro-inflammatory IL-8 binder) or the affitin protein gene (EpCAM tumor antigen binder) and the sequence for the peptide tag Cmyc or Flag were inserted into the constructs. The latter are an important tool in biochemistry, since they allow, while attached to a protein in combination with specific antibodies, the detection, purification, and characterization of recombinant proteins. The constructs were expressed in L. lactis bacteria, which are a suitable vectors for the oral delivery of the mentioned therapeutic proteins due to their long-term recognized safety and simple genetic modification. The expression of individual proteins EVA-Cmyc, AFFI-Cmyc, EVA-Flag, and AFFI-Flag or their combinations was confirmed by using peptide tags, their presence was proved in cell lysates and also on the surface of L. lactis bacteria, with the extent of surface presentation varying, depending on the type of protein, introduced peptide tag and also depending on whether the protein was expressed individually or simultaneously with other proteins. Finally, we demonstrated that bacteria with surface-displayed evasins and affitins are functional, i.e., capable of binding to IL-8 and the EpCAM receptor. Due to the general disapproval of GMOs, we separated the expressed proteins from the producer cells, attached them non-covalently to wild strains of bacteria that were not genetically modified, and proved their surface presentation. The entire evaluation of proteins was performed on the basis of peptide tags and demonstrated that their use is a suitable, affordable and easy way to detect and differentiate expressed proteins, which was also the basic purpose of the master's thesis. We have found that although peptide tags sterically interfere with the binding sites of proteins and affect their functionality, the advantages of their use outweigh the mentioned disadvantages.