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Utveckling av reningsmetod för cytokrom c-Id1 från Ideonella dechloratans
Karlstad University, Faculty of Technology and Science. (Kloratgruppen)
2012 (Swedish)Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesisAlternative title
Otimization of a Purification Method for Cytochrome c-Id1 from Ideonella dechloratans (English)
Abstract [sv]

Syftet med arbetet var att utveckla en metod för att rena fram cytokrom c-Id1 ur periplasma från Ideonella dechloratans. Inledningsvis undersöktes proteinets stabilitet vid låga pH. Flera olika kromatografiska metoder provades under arbetets gång. Till de inledande experimenten användes flow-through fraktioner från rening av annat protein som startmaterial. Senare försök gjordes på periplasma från I. dechloratans.

Stabilitetsundersökningen gjordes genom att proteinlösningens spektrum registrerades. Lösningen reducerades sedan med ditionit och ett nytt spektrum registrerades. Ett differensspektrum beräknades och absorbansmaximum vid 550 nm användes som mått på mängden cytokrom c-Id1. Undersökningen visade att proteinet är stabilt vid pH ner till 3.0.

Första försöken med respektive kromatografisk metod, förutom gelfiltrering, gick ut på att undersöka om proteinet kunde adsorberas till kolonnen eller inte. När proteinet adsorberats till kolonnen ändrades målet med försöken till att försöka eluera det så rent som möjligt.

Den slutliga reningsmetoden innehåller tre kromatografiska steg, varav ett behöver optimeras ytterligare för att ge rent protein. Första steget i reningen är katjonbyteskromatografi. Det andra stegetär hydrofob interaktionskromatografi och det sista steget är anjonbyteskromatografi. Efter reningen fanns små mängder föroreningar kvar. Två oönskade proteiner, båda i liten mängd fanns kvar i provet. För att få proteinet helt rent krävs ytterligare optimering av reningsmetoden. Störst potential till förbättring finns troligen i steget med anjonbytaren.

Misstankar om att proteinet lätt dimeriserar eller reagerar på annat vis under lagring har funnits under hela arbetet. Resultat från bland annat gelfiltrering har indikerat att så är fallet. Arbetet har inte gettsvar på om cytokrom c-Id1 förändras med tiden eller inte.

Bestämning av totala mängden protein samt mängden cytokrom c-Id1 efter reningen gjordes. Dessa analyser visar att 69 μg protein, varav 36 μg cytokrom c-Id1 återstod efter rening från en odling på fyra liter. Halten cytokrom c-Id1 var alltså 52 %.

Abstract [en]

The aim of this study was to develop a method to purify cytochrome c-Id1 from periplasmic extract of Ideonella dechloratans. First the stability of the protein at low pH was investigated. Several different chromatographic methods were tested during the study. In the first experiments the starting material was flow through fractions from purification of another protein. Later experiments were performed with periplasmic extract from I. dechloratans.

The stability determination was made by recording a spectrum from the protein solution. Proteins were then reduced, by dithionite, and a new spectrum was recorded. A difference spectrum was calculated and the absorbance at 550 nm was used as a measure of the amount of cytochrome c-Id1. The investigation showed that the protein is stable at pH 3.0 and higher.

The first experiments with each chromatographic method, except gel filtration, were made in order to see whether the protein could be adsorbed to the column or not. When the protein had been adsorbed to the column the target of the experiments changed. The target was then to elude the protein as pure as possible.

The resulting purification method contains three chromatographic steps. One of these needs to be further optimized in order to give pure protein. The first step is cation exchange chromatography. The second step is hydrophobic interaction chromatography and the last step is anion exchange chromatography. After purification with this method, small amounts of contaminants were still present. Two unwanted proteins are present, in small amounts. In order to get the protein completely pure further optimization of the method is required. The biggest optimization potential is probably in the anion exchange chromatography step.

During this work there have been some indications that the protein easily dimerizes or reacts in some other way upon storage. Results from for example gel filtration have indicated that so is the case. This work has not given an answer to whether cytochrome c-Id1 changes upon storage or not.

The total amount of protein as well as the amount of cytochrome c-Id1 after the purification was determined. The analyses showed that the total amount of protein was 69 μg and the amount of cytochrome c-Id1 was 36 μg from four liters of culture. This means that the cytochrome c-Id1 content afterthe purification was 52 %.

Place, publisher, year, edition, pages
2012. , 31 p.
Keyword [sv]
Ideonella dechloratans, proteinrening, cytokrom c-Id1
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:kau:diva-13604Local ID: KEM-31OAI: oai:DiVA.org:kau-13604DiVA: diva2:533971
Subject / course
Chemistry
Uppsok
Physics, Chemistry, Mathematics
Supervisors
Examiners
Available from: 2012-06-21 Created: 2012-06-12 Last updated: 2012-06-21Bibliographically approved

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