Finding a stabilized sequence for reconstructed Icosahedra Lumazine Synthase by random mutagenesis and Fluorescence-Activated Cell Sorting
(2019) KEMR33 20191Department of Chemistry
- Abstract
- Lumazine Synthase forms pentamers, C5 in some organism and icodahedra, Ih in other. The aim of this project is to investigate which evolutionary steps could have led the pentamers to assembly into icosahedral capsids. Therefore prevoius work has been performed to reconstruct ancestral sequences for the pentameric and icosahedral forms of Lumazine Synthase. The icosahedral sequence had been expressed and shown to give insoluble expression. The work in this thesis has therefore been done to, firstly, try to purify and refold the protein from inclusion bodies and secondly, try to improve the sequence to give soluble expression.
Expression of the protein resulted in the protein in inclusion bodies. The protein could be purified from the... (More) - Lumazine Synthase forms pentamers, C5 in some organism and icodahedra, Ih in other. The aim of this project is to investigate which evolutionary steps could have led the pentamers to assembly into icosahedral capsids. Therefore prevoius work has been performed to reconstruct ancestral sequences for the pentameric and icosahedral forms of Lumazine Synthase. The icosahedral sequence had been expressed and shown to give insoluble expression. The work in this thesis has therefore been done to, firstly, try to purify and refold the protein from inclusion bodies and secondly, try to improve the sequence to give soluble expression.
Expression of the protein resulted in the protein in inclusion bodies. The protein could be purified from the inclusion bodies by guanidine·HCl extraction and size exclusion chromatography. However it was not possible to refold the protein into native structure by dialysis to remove the denturant, guanidine·HCl.
To improve the sequence to give stable expression, a stability assay system was used in combination with random mutagenesis of the sequence. The stability assay system consist of two reporter proteins, red fluorescent tagRFP and green fluorescent sf -GFP. Expression of the protein is monitored by red fluorescence by tag RFP fused to the protein and the stability of the expressed protein is monitored by sf -GFP expressed under the control of stress activated DnaK promoter. The random mutagen- esis was performed by error prone PCR to produce fragments spanning the sequence of Lumazine Synthase. The created fragments were then used as megaprimers to amplify the whole plasmid containing the protein with tagRFP and the stability assay system. (Less) - Popular Abstract (Swedish)
- Proteiner är de små byggstenar som bygger upp nästan allt omkring oss. Proteiner bygger upp våra kroppar och utför olika funktioner som är livsviktiga för alla djur, växter och andra organismer. Byggstenarna kan ha olika former och utseende och de har olika funktioner. Det finns proteiner som hjälper till att bryta ner maten vi äter, proteiner som bygger upp våra muskler och massor av andra proteiner som hjälper till med olika kemiska reaktioner. Proteinerna består av långa kedjor av aminosyror som sitter ihop i en viss ordning, en viss sekvens. Genom evolutionens gång har proteinernas sekvenser förändrats och utvecklats till de varianter som finns i olika organismer idag. Många proteiner är lite annorlunda beroende på vilken organism man... (More)
- Proteiner är de små byggstenar som bygger upp nästan allt omkring oss. Proteiner bygger upp våra kroppar och utför olika funktioner som är livsviktiga för alla djur, växter och andra organismer. Byggstenarna kan ha olika former och utseende och de har olika funktioner. Det finns proteiner som hjälper till att bryta ner maten vi äter, proteiner som bygger upp våra muskler och massor av andra proteiner som hjälper till med olika kemiska reaktioner. Proteinerna består av långa kedjor av aminosyror som sitter ihop i en viss ordning, en viss sekvens. Genom evolutionens gång har proteinernas sekvenser förändrats och utvecklats till de varianter som finns i olika organismer idag. Många proteiner är lite annorlunda beroende på vilken organism man undersöker.
Det protein som detta arbete undersöker heter Lumazine Synthase och det bildar tre olika varianter i olika organsimer. I svampar och arkéer bildar 5 proteinenheter, som sitter i en cirkel tillsammans, så kallade pentamerer av proteinet. I de flesta bakterier bildar 12 stycken pentamerer tillsammans en sfärisk struktur. Ungefär som rutorna på en fotboll sitter de 12 pentamererna sida vid sida och bildar en rund kapsel. I vissa organismer sitter två pentamerer ihop som en sandwich. Det är intressant att det bara är i vissa organismer som enheterna bildar kapslar medan de i andra organismer bara bildar pentamerer eller dubbla pentamerer. Därför vill man undersöka hur det kan ha gått till när evolutionen har gjort så att det finns dessa varianter. Det måste ha skett några förändringar i strukturen som gjorde att pentamererna kunde sitta ihop i varandra och bilda kapslar. Därför har man räknat ut vilken sekvens den första Lumazine Synthase som bildade en kapsel kan ha haft.
Det som ställde till med ett problem var att sekvensen man räknat fram inte lyckades bilda ett stabilt protein. Därför ville man försöka ändra på sekvensen litegrann för att få ett lite mer stabilt protein. Detta kan man göra genom att driva en slags evolution på liten skala genom att göra slumpmässiga förändringar i aminosyrasekvensen och sen välja ut varianter som fungerar bättre. Tanken är då att någon av de slumpmässiga förändringarna ska ge en variant som bildar ett mer stabilt protein vilket man sen kan använda för att undersöka vidare. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8984809
- author
- Olsson, Caroline LU
- supervisor
- organization
- course
- KEMR33 20191
- year
- 2019
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Icosahedra, Assembly, Lumazine Synthase, Fluorescence-Activated Cell Sorting, FACS, Directed evolution, Random mutagenesis, Stability, Structure, Biochemistry, Biokemi
- language
- English
- id
- 8984809
- date added to LUP
- 2019-07-04 15:57:50
- date last changed
- 2019-07-04 15:57:50
@misc{8984809,
abstract = {{Lumazine Synthase forms pentamers, C5 in some organism and icodahedra, Ih in other. The aim of this project is to investigate which evolutionary steps could have led the pentamers to assembly into icosahedral capsids. Therefore prevoius work has been performed to reconstruct ancestral sequences for the pentameric and icosahedral forms of Lumazine Synthase. The icosahedral sequence had been expressed and shown to give insoluble expression. The work in this thesis has therefore been done to, firstly, try to purify and refold the protein from inclusion bodies and secondly, try to improve the sequence to give soluble expression.
Expression of the protein resulted in the protein in inclusion bodies. The protein could be purified from the inclusion bodies by guanidine·HCl extraction and size exclusion chromatography. However it was not possible to refold the protein into native structure by dialysis to remove the denturant, guanidine·HCl.
To improve the sequence to give stable expression, a stability assay system was used in combination with random mutagenesis of the sequence. The stability assay system consist of two reporter proteins, red fluorescent tagRFP and green fluorescent sf -GFP. Expression of the protein is monitored by red fluorescence by tag RFP fused to the protein and the stability of the expressed protein is monitored by sf -GFP expressed under the control of stress activated DnaK promoter. The random mutagen- esis was performed by error prone PCR to produce fragments spanning the sequence of Lumazine Synthase. The created fragments were then used as megaprimers to amplify the whole plasmid containing the protein with tagRFP and the stability assay system.}},
author = {{Olsson, Caroline}},
language = {{eng}},
note = {{Student Paper}},
title = {{Finding a stabilized sequence for reconstructed Icosahedra Lumazine Synthase by random mutagenesis and Fluorescence-Activated Cell Sorting}},
year = {{2019}},
}