Molekulsko kloniranje in izražanje rekombinantne indolamin 2,3-dioksigenaze 1 v bakteriji Escherichia coli

Petrač, Mirjam (author), Bratkovič, Tomaž (mentor)

Temelj magistrske naloge je bila priprava ekspresijske kasete, molekulsko kloniranje, izražanje ter izolacija in čiščenje humanega proteina indolamin 2,3-dioksigenaze 1 (IDO1). Rekombinanten encim bo omogočil razvoj in vrednotenje novih zaviralcev encima IDO1, potencialno uporabnih za zdravljenje nekaterih rakavih bolezni. IDO1 je normalno prisoten encim v človeških celicah. Njegova naloga je indukcija primarne poti katabolizma triptofana. Pri tem nastanejo presnovki, med katerimi so nekateri tudi toksični. Skozi pot metabolizma triptofana in nastanek kinurenina, ki je ligand za arilni ogljikovodikov receptorjev (AhR) pride do prikritja tumorja imunskemu sistemu. Tako je omogočena njegova rast in razvoj. Rekombinantni humani IDO1 (rhIDO1) smo izražali v celicah Escherichia coli NiCo21. V vektor pET-28 smo vstavili gen za IDO1, ki smo mu na 5'-konec pripeli nukleotidno zaporedje, kodirajoče heksahistidinski rep in peptidno zaporedje, ki ga selektivno cepi trombin. Plazmid smo s toplotnim šokom vstavili v ekspresijski bakterijski sev. Celice smo namnožili in z dodatkom induktorja IPTG inducirali izražanje rhIDO1. Skozi poizkuse smo iskali najustreznejše pogoje (temperatura, čas indukcije, koncentracija glukoze, vrsta gojišča…) s ciljem optimizacije izražanja z vidika kakovosti in količine produkta. Za čiščenje smo uporabili večstopenjski kromatografski proces (kovinsko-kelatno afinitetno kromatografijo, gelsko filtracijo in kationsko izmenjevalno kromatografijo). Čistoto produkta smo med izolacijo spremljali s polialkrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata. Koncentracijo produkta smo ocenili spektrofotometrično in z metodo po Bradfordu. Na koncu smo aktivnost proteina preverili z biokemijskim testiranjem. Uspešno smo izrazili in izolirali rhIDO1. Čeprav je produkt vseboval določen delež nečistot in je izkazoval nižjo aktivnost v primerjavi s komercialnim standardom, ga kljub temu lahko uporabljamo za vrednotenje potencialnih zaviralcev rhIDO1 v encimskih testih.

• Language: Slovenian
• Keywords: indolamin 2, 3-dioksigenaza 1, Escherichia coli, molekulsko kloniranje, izražanje, izolacija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-114855
• Publication date in RUL: 20.03.2020

Secondary language

• Language: English
• Title: Cloning and expression of recombinant indolamine 2,3-dioxygenase 1 in Escherichia coli

Abstract

The basis of my master’s degree was to prepare an expression cassette, molecular cloning and isolation, and purification of human indoleamine 2,3-dioxygenase 1 (IDO1). The recombinant enzyme will enable the evolution and development of new inhibitors of IDO1, which might potentially be used in cancer treatment. IDO1 is a normally presented enzyme in human cells. It catalyses the primary path in the catabolism of tryptophan. During the process, some toxic metabolites are synthetized. Through the metabolism of tryptophan and the formation of kynurenine, which is a ligand of aryl hydrocarbon receptors (AhR), tumors are hidden from our immune system. This facilitates their growth and development. Recombinant human IDO1 (rhIDO1) was expressed in Escherichia coli NiCo21 cells. The gene for IDO1 was inserted into vector pET_28. On 5'-side of gene, we inserted a nucleotide sequence for hexahystidine tail and a peptide sequence, which can be cleaved with thrombin. We inserted the plasmid into the expression system by the means of heat shock. We multiplied cells which received the insert and added Isopropyl β-D-1-thiogalactopyranoside (IPTG), which induced the expression of rhIDO1. Our experiments focused on a search for the most appropriate conditions (temperature, time of induction, concentration of glucose, type of medium …) with the aim to optimize the expression and improve the quality and quantity of product. For purification we used a multistage chromatography process (immobilized metal affinity chromatography, size-exclusion chromatography and ion-exchange chromatography). The purity of product was monitored with polyacrylamide gel electrophoresis. The concentration of product was measured with spectrophotometry and the Bradford method. At the end we tested activity of IDO1 with biochemical testing. The expression of rhIDO1 was successful. Although the product has some impurities and was less active than the commercial standard, we can still use it for evaluation of potential inhibitors of rhIDO1 in enzyme testing.

• Keywords: indoleamine 2, 3, .dioxigenase, Escherichia coli, molecular cloning, expression, isolation