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Cloning, Purification and Characterization of Ribonucleotide Reductases from Pseudomonas aeruginosa and Chlamydia trachomatis

Hansson, Ragnar LU (2018) KEMR13 20172
Department of Chemistry
Abstract
Ribonucleotide reductases (RNRs) are important enzymes that convert the building blocks of RNA (NTPs) into building blocks of DNA (dNTPs). dNTPs are used for synthesis of DNA as well as repair of mutated and damaged DNA. These cellular processes are critical to an organism. That is why these processes are found in various organisms. Since these processes are so essential, their allosteric regulation is tightly controlled to maintain the conversions. They are divided into three main classes that are mainly determined by the different radical mechanisms of these enzymes. Investigations of RNRs from different organisms have led to structural insights such as crystal structures. This has made deeper understanding of these important enzyme’s... (More)
Ribonucleotide reductases (RNRs) are important enzymes that convert the building blocks of RNA (NTPs) into building blocks of DNA (dNTPs). dNTPs are used for synthesis of DNA as well as repair of mutated and damaged DNA. These cellular processes are critical to an organism. That is why these processes are found in various organisms. Since these processes are so essential, their allosteric regulation is tightly controlled to maintain the conversions. They are divided into three main classes that are mainly determined by the different radical mechanisms of these enzymes. Investigations of RNRs from different organisms have led to structural insights such as crystal structures. This has made deeper understanding of these important enzyme’s allosteric regulation, catalytical mechanisms and how they are related to each other from an evolutionary perspective. The structural understanding of these enzymes could also lead to drug discoveries since they are essential, which makes them to be good candidates as target proteins.
The most common RNR is built up of two proteins that together catalyze the reaction. One domain is the so called large subunit α2 (historically also known as R1) where the main catalysis takes place and the other is the so called small subunit β2 (historically also known as R2) where the free radical formation takes place.
In the first part of this work the subunits NrdD (large) and NrdG (small) from class III RNR from Pseudomonas aeruginosa were investigated. These two proteins have previously resulted in insoluble inclusion bodies and in this work they were attached by solubility tags found in the three vectors pETM41/pETM50/pETM60. The constructs were then expressed to see if the proteins were soluble or not. Further studies need to be done to determine whether the solubility tags from the three different vectors improved the solubility or not.
In the second part of this work, the aerobic RNR class I enzyme from Chlamydia trachomatis was purified and later investigated by electron microscopy (EM) and cryo-electron microscopy (Cryo-EM) in a step to get a deeper insight in their structure and function. The results from the EM looked promising, although the Cryo-EM data needs to be analyzed to draw any conclusions. DLS-analysis and crystallization trials remain to be done in the future in pursuit of solving this structure. (Less)
Popular Abstract (Swedish)
Ribonukleotidreduktaser (RNR) är livsviktiga enzymer som är involverade i nästan alla levande organismer. Enzymernas uppgift är att omvandla ribonukleotider (NTPer) till deoxyribonukleotider (dNTPer), byggstenar som är essentiella för såväl DNA-syntes som DNA-reparation. Enzymernas aktivitet baseras på en radikal-mekanism som skiljer sig, vilket gör att RNRerna är uppdelade i tre olika klasser. Utöver detta är enzymerna ibland även uppdelade i underklasser men dessa skiljelinjer är ibland en definitionsfråga som inte alltid är antingen svart eller vit.
Dessa livsviktiga enzymer gör att karakterisering av dem är viktigt. Dels för att öka förståelsen över deras aktivitet, men också för att förstå korrelationen mellan olika organismer och... (More)
Ribonukleotidreduktaser (RNR) är livsviktiga enzymer som är involverade i nästan alla levande organismer. Enzymernas uppgift är att omvandla ribonukleotider (NTPer) till deoxyribonukleotider (dNTPer), byggstenar som är essentiella för såväl DNA-syntes som DNA-reparation. Enzymernas aktivitet baseras på en radikal-mekanism som skiljer sig, vilket gör att RNRerna är uppdelade i tre olika klasser. Utöver detta är enzymerna ibland även uppdelade i underklasser men dessa skiljelinjer är ibland en definitionsfråga som inte alltid är antingen svart eller vit.
Dessa livsviktiga enzymer gör att karakterisering av dem är viktigt. Dels för att öka förståelsen över deras aktivitet, men också för att förstå korrelationen mellan olika organismer och hur evolutionen påverkat utvecklingen från början till nu. Detta kan i sin tur leda till att man kan förstå enzymernas involvering I olika typer av sjukdomar och därifrån genomföra olika mikrobiologiska modifieringar och analyser vars upptäckter förhoppningsvis kan användas för att upptäcka läkemedel.
I första delen av detta arbete så studerades RNR klass III proteinerna NrdD och NrdG från bakterien Pseudomonas aeruginosa. Tidigare försök att studera dessa två proteiner har genererat svårlösliga inklusionskroppar vilket gjort att man försökt klona in generna som uttrycker dessa två proteiner i plasmider som före det inklonade proteinet uttrycker proteiner som är kända för att öka lösligheten för proteiner vars uttryck uppvisar liknande tendenser som i detta fall. I arbetet användes tre olika plasmider (pETM41/pETM50/petM60) som alla tre har olika taggar som alla är kända för att öka lösligheten. Dessa tre plasmider innehåller även alla tre His-taggar framför sina respektive löslighetstaggar som gör det enkelt att rena fram det önskvärda proteinet i fråga med hjälp av affinitetskromatografiupprening. Fem av dessa sex kloningar fungerade, men däremot inte det sjätte trots upprepade försök. Bara en utav de fem lyckade kloningarna genererade överexpression, vilket betyder att de fyra misslyckade överexpressionerna måste testas fler gånger.
I den andra delen delen av detta arbete så upprenades aeroba RNR klass I proteiner från Chlamydia trachomatis som sedan undersöktes med hjälp av elektronmikroskopi (EM) och kryoelektronmikroskopi (CryoEM). EM undersökningarna såg lovande ut, däremot fanns det tyvärr ingen tid att analysera data från CryoEM undersökningen. Tidsbristen gjorde även att undersökningar med dynamisk ljusspridning (DLS) och kristalliseringsförsök tyvärr uteblev. (Less)
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author
Hansson, Ragnar LU
supervisor
organization
course
KEMR13 20172
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Ribonucleotide Reductases, RNR, RNRs, Cryo-EM, dNTPs, DLS, biochemistry, biokemi
language
English
id
8957597
date added to LUP
2018-09-12 11:19:04
date last changed
2018-09-12 11:19:04
@misc{8957597,
  abstract     = {Ribonucleotide reductases (RNRs) are important enzymes that convert the building blocks of RNA (NTPs) into building blocks of DNA (dNTPs). dNTPs are used for synthesis of DNA as well as repair of mutated and damaged DNA. These cellular processes are critical to an organism. That is why these processes are found in various organisms. Since these processes are so essential, their allosteric regulation is tightly controlled to maintain the conversions. They are divided into three main classes that are mainly determined by the different radical mechanisms of these enzymes. Investigations of RNRs from different organisms have led to structural insights such as crystal structures. This has made deeper understanding of these important enzyme’s allosteric regulation, catalytical mechanisms and how they are related to each other from an evolutionary perspective. The structural understanding of these enzymes could also lead to drug discoveries since they are essential, which makes them to be good candidates as target proteins.
The most common RNR is built up of two proteins that together catalyze the reaction. One domain is the so called large subunit α2 (historically also known as R1) where the main catalysis takes place and the other is the so called small subunit β2 (historically also known as R2) where the free radical formation takes place.
In the first part of this work the subunits NrdD (large) and NrdG (small) from class III RNR from Pseudomonas aeruginosa were investigated. These two proteins have previously resulted in insoluble inclusion bodies and in this work they were attached by solubility tags found in the three vectors pETM41/pETM50/pETM60. The constructs were then expressed to see if the proteins were soluble or not. Further studies need to be done to determine whether the solubility tags from the three different vectors improved the solubility or not.
In the second part of this work, the aerobic RNR class I enzyme from Chlamydia trachomatis was purified and later investigated by electron microscopy (EM) and cryo-electron microscopy (Cryo-EM) in a step to get a deeper insight in their structure and function. The results from the EM looked promising, although the Cryo-EM data needs to be analyzed to draw any conclusions. DLS-analysis and crystallization trials remain to be done in the future in pursuit of solving this structure.},
  author       = {Hansson, Ragnar},
  keyword      = {Ribonucleotide Reductases,RNR,RNRs,Cryo-EM,dNTPs,DLS,biochemistry,biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Cloning, Purification and Characterization of Ribonucleotide Reductases from Pseudomonas aeruginosa and Chlamydia trachomatis},
  year         = {2018},
}