Priprava rdečega fluorescenčnega proteina z N-končnim imunoglobulin-vezavnim peptidom

Jašovič, Vita (author), Bratkovič, Tomaž (mentor), Kruljec, Nika (co-mentor)

V celični in molekularni biologiji pogosto uporabljamo poročevalne proteine, s katerimi označujemo strukture v celicah ali tkivih. Gre pravzaprav za fluorokrome, pri katerih je ob osvetljevanju značilen pojav fluorescence, ko molekula absorbira svetlobo določene valovne dolžine in nato izseva svetlobo nižje energije, torej daljše valovne dolžine. Za označevanje celičnih struktur s poročevalnimi proteini moramo običajno v organizem vnesti njihov genski zapis. Rdeči fluorescenčni protein (RFP) v vzbujenem stanju fluorescira rdeče-oranžno, z maksimalnim vzbujanjem pri 558 nm ter emisijskim maksimumom pri 583 nm. Zaradi globljega prodiranja rdeče svetlobe skozi tkiva te fluorescenčne proteine uporabljamo pri mikroskopiji debelejših tkivnih vzorcev. V okviru diplomske naloge smo kot poročevalski protein uporabili RFP in mu na N-konec pripeli imunoglobulin-vezavni peptid min19Fc_Q6D z aminokislinskim zaporedjem GSYWYDVWF. Peptidi bi lahko postali alternativa bakterijskim imunoglobulin-vezavnim proteinom, ki jih uporabljamo kot specifične ligande protiteles (npr. pri afinitetni kromatografiji za čiščenje monoklonskih protiteles iz procesne tekočine), saj imajo le-ti številne prednosti. Z afinitetnim ligandom min19Fc_Q6D, ki izkazuje afniteto do regije Fc protiteles IgG, smo tvorili fuzijski protein min19Fc_Q6D-RFP, pri katerem smo lahko zaradi poročevalnega proteina z UV-detekcijo spremljali njegovo izražanje ter skušali vezavo na IgG vrednotiti s fluorescenčnoimunskim testom (FLISA). Gen za fuzijski protein smo pripravili z verižno reakcijo s polimerazo in ga vstavili v ekspresijski vektor. Rekombinantni protein smo uspešno izrazili v bakteriji Escherichia coli NiCo21. V nadaljevanju smo ga s pomočjo kovinsko-kelatne afinitetne kromatografije ter nadaljnjega razsoljevanja z gelsko izključitveno kromatografijo ustrezno očistili, izolirali ter ga pripravili za vrednotenje vezave na regijo Fc protiteles IgG. Interakcije s protitelesi nismo uspeli potrditi. Možne razlage za to so: i) RFP sterično ovira interakcijo med peptidom min19Fc_Q6D in protitelesi, ii) interakcija termodinamsko ni ugodna zaradi znižanja entropije, do katere bi prišlo ob vezavi, ali iii) test FLISA ni dovolj občutljiv, saj signal izhaja zgolj iz monosloja molekul na dnu vdolbinic mikrotitrske ploščice.

• Language: Slovenian
• Keywords: rdeči fluorescenčni protein, fuzijski protein, min19Fc_Q6D, peptid, protitelesa
• Work type: Bachelor thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-119189
• Publication date in RUL: 04.09.2020

Secondary language

• Language: English
• Title: Preparation of a red fluorescent protein with the N-terminal immunoglobulin-binding peptide

Abstract

In cell and molecular biology, reporter proteins are commonly used for labeling cell or tissue structures. Reporter proteins are often fluorochromes, which are characterized by the appearance of fluorescence. When illuminated, the molecule absorbs light of a certain wavelength and then emits light of lower energy, i.e. longer wavelengths. To label cell structures with reporter proteins, one usually needs to introduce their genetic record into the organism. The excited red fluorescent protein (RFP) fluoresces red-orange, with a maximum excitation at 558 nm and an emission maximum at 583 nm. Due to the deeper penetration of red light through tissues, red fluorescent proteins are used in the microscopy of thicker samples. As part of the thesis, we used RFP as a reporter protein and attached the immunoglobulin binding peptide min19Fc_Q6D, with the amino acid sequence GSYWYDVWF, to its N-terminus. Peptides could become an alternative to the bacterial immunoglobulin-binding proteins used as specific ligands for antibodies (e.g., in affinity chromatography to purify monoclonal antibodies from the process fluid), as they have many advantages. We chose the peptide ligand min19Fc_6QD as it displays affinity to the Fc region of immunoglobulins G, to form the fusion protein min19Fc_Q6D-RFP. We were able to monitor its expression due to the reporter protein. We attempted to evaluate its binding to IgGs by fluorescence-linked immunosorbent assay (FLISA). The gene for the fusion protein was constructed by polymerase chain reaction and inserted into the expression vector. We successfully expressed the recombinant protein in Escherichia coli NiCo21 cells, and purified it by immobilized metal affinity and size exclusion chromatography. We tried to evaluate its binding to the Fc region of IgG antibodies, however, no interaction could be detected. There are several possible explanations for that: i) RFP might sterically hinder the min19Fc_Q6D peptide:antibody interaction; ii) the interaction is thermodynamically disfavoured due to high entropic cost upon binding; and/or iii) the FLISA assay is not sensitive enough to detect binding as the signal is only produced on a molecular monolayer at the bottom of microtiter plate wells.

• Keywords: red fluorescent protein, fusion protein, min19Fc_Q6D, peptide, antibodies