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Integration of Aqueous Two-Phase Systems into Recovery Processes for Biomolecules

Kepka, Cecilia LU (2004)
Abstract (Swedish)
Popular Abstract in Swedish

Människans kropp består av celler som byggs upp av komponenter som DNA, proteiner, lipider och polysackarider. I DNA finns den genetiska informationen lagrad som gör att kroppen kan fungera. Informationen i DNA omvandlas till proteiner som deltager i cellens biokemiska processer. DNA finns i olika former, dels genomiskt DNA och dels en mindre cirkulär form som kallas plasmid DNA. Plasmid DNA från bakterier används inom gentekniken där plasmiden kan förses med önskvärda egenskaper. Plasmid DNA kan återföras i celler för att producera proteiner med speciella egenskaper. Stort intresse finns idag för användning av plasmid DNA för att kunna bota olika sjukdomar med genterapi.



I... (More)
Popular Abstract in Swedish

Människans kropp består av celler som byggs upp av komponenter som DNA, proteiner, lipider och polysackarider. I DNA finns den genetiska informationen lagrad som gör att kroppen kan fungera. Informationen i DNA omvandlas till proteiner som deltager i cellens biokemiska processer. DNA finns i olika former, dels genomiskt DNA och dels en mindre cirkulär form som kallas plasmid DNA. Plasmid DNA från bakterier används inom gentekniken där plasmiden kan förses med önskvärda egenskaper. Plasmid DNA kan återföras i celler för att producera proteiner med speciella egenskaper. Stort intresse finns idag för användning av plasmid DNA för att kunna bota olika sjukdomar med genterapi.



I dagens biotekniska industri används bakterier för att tillverka proteiner för olika användningsområden. Om man odlar en gentekniskt modifierad bakterie under lämpliga förhållanden, dvs vid rätt temperatur och med näring, så producerar bakterien önskat protein eller plasmid. Bra uppreningsmetoder behövs för att isolera det önskvärda proteinet eller plasmiden. Olika krav på renhet ställs beroende på användningsområde. Om ett protein ska ingå i ett läkemedel krävs en hög grad av renhet. Men om proteinet är ett enzym som exempelvis ska ingå i tvättmedel krävs inte en lika ren produkt.



De största kostnaderna idag för framställning av ett läkemedel baserat på protein är relaterade till uppreningen av proteinet. Därför är det viktigt att utveckla och studera nya reningsmetoder, vilket är ämnet för denna avhandling. För en reningsmetod i bioteknisk industri krävs att den ska vara kostnadseffektiv, snabb och kunna användas tillsammans men andra reningstekniker. En produkt blir ofta inte upprenad i ett steg utan flera olika reningssteg krävs. En effektiv separationsmetod för proteiner och plasmider är vattenhaltiga tvåfassystem. Dessa består av polymerer i vatten som efter blandning separerar i två faser, vilka båda innehåller en hög andel vatten. Tvåfassystem är lämpliga för biologiska material eftersom dessa ofta är känsliga. Biologiska produkter, som proteiner och plasmider, kan fördelas mellan faserna beroende på produkternas egenskaper. Det är oftast önskvärt att produkten fördelas till den övre fasen medan föroreningar fördelas till den nedre fasen. Systemen kan modifieras för att styra produkten till önskad fas. Ett protein kan modifieras med genteknik för att få den önskade fördelningen. I avhandlingen visas hur ett protein kan förändras med en sekvens av aminosyror för effektiv upprening. Tvåfassystemen kan optimeras i liten skala och sedan skalas upp för industriell användning med bibehållen effektivitet.



Olika tvåfassystem har utvecklats för rening av proteiner samt plasmid DNA från bakterieceller. För att kunna kombinera ihop olika separationstekniker är det viktigt att tvåfassystemet har egenskaper som gör detta möjligt. I avhandlingen visas hur plasmid DNA kan upprenas för användning i läkemedel genom att integrera olika tekniker till en effektiv process. (Less)
Abstract
The main scope of this thesis has been to study and develop new purification methods and also to further develop existing methods for purification to enhance their efficacy. Important parameters in the development of the methods are that they should be suitable for scale-up and possible to be integrated into recovery processes.



The studies in this thesis describe that a hydrophobic peptide tag can be fused to the enzyme cutinase in order to enhance the purification in aqueous two-phase systems. The addition of a hydrophobic peptide tag composed of four tryptophans and prolines to cutinase resulted in increased partitioning in a two-phase system. One aim of this thesis has been to develop a system composed of a... (More)
The main scope of this thesis has been to study and develop new purification methods and also to further develop existing methods for purification to enhance their efficacy. Important parameters in the development of the methods are that they should be suitable for scale-up and possible to be integrated into recovery processes.



The studies in this thesis describe that a hydrophobic peptide tag can be fused to the enzyme cutinase in order to enhance the purification in aqueous two-phase systems. The addition of a hydrophobic peptide tag composed of four tryptophans and prolines to cutinase resulted in increased partitioning in a two-phase system. One aim of this thesis has been to develop a system composed of a thermoseparating polymer and a low cost starch for extraction of the recombinant cutinase from E. coli homogenate. The developed system was scaled up to 400 l and a disk-stack centrifugal separator was adapted for rapid separation of the two-phase system. The target protein was recovered in a water phase with an overall yield of 71%.The recombinant tagged cutinase was also extracted in a two-phase system composed of PEG and salt. The target protein was recovered with high yield in the PEG top phase. The top phase was further interfaced with hydrophobic interaction chromatography and the target protein was obtained in a polymer free phase with a total recovery of 83% after the two extraction steps.



The mechanisms for partitioning of plasmid DNA in polymer/polymer systems was determined. A system composed of the thermoseparating EOPO polymer and dextran was developed for purification of plasmid DNA . The plasmid DNA from a desalted lysate could be completely recovered in the EOPO top phase. By increasing the temperature of the top phase, phase separation was induced and the plasmid DNA was obtained in a water phase. The system was effective in concentrating the sample volume with concomitant removal of contaminants e.g. RNA and proteins. The thermoseparating system was also integrated with membrane filtration and restricted access chromatography resulting in a three step process for purification of plasmid DNA. Plasmid DNA was effectively purified from contaminants such as RNA and proteins and was recovered with a total yield of 69% in this process. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Professor Lydiatt, Andrew, Millipore Bioprocessing Ltd, Consett, UK
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Proteins, proteins, plasmid DNA, large-scale, protein purification, integration, Expression, aqueous two-phase systems, enzymology, Proteiner, enzymologi
pages
143 pages
publisher
Folke Tjerneld, Department of Biochemistry, Lund University
defense location
Sal B, Chemical Center, Lund
defense date
2004-10-29 10:15
ISBN
91-628-6222-7
language
English
LU publication?
yes
id
3ef7fa69-c6a7-4a99-85d3-c387569ad1ec (old id 467462)
date added to LUP
2007-10-13 14:14:42
date last changed
2016-09-19 08:45:07
@misc{3ef7fa69-c6a7-4a99-85d3-c387569ad1ec,
  abstract     = {The main scope of this thesis has been to study and develop new purification methods and also to further develop existing methods for purification to enhance their efficacy. Important parameters in the development of the methods are that they should be suitable for scale-up and possible to be integrated into recovery processes.<br/><br>
<br/><br>
The studies in this thesis describe that a hydrophobic peptide tag can be fused to the enzyme cutinase in order to enhance the purification in aqueous two-phase systems. The addition of a hydrophobic peptide tag composed of four tryptophans and prolines to cutinase resulted in increased partitioning in a two-phase system. One aim of this thesis has been to develop a system composed of a thermoseparating polymer and a low cost starch for extraction of the recombinant cutinase from E. coli homogenate. The developed system was scaled up to 400 l and a disk-stack centrifugal separator was adapted for rapid separation of the two-phase system. The target protein was recovered in a water phase with an overall yield of 71%.The recombinant tagged cutinase was also extracted in a two-phase system composed of PEG and salt. The target protein was recovered with high yield in the PEG top phase. The top phase was further interfaced with hydrophobic interaction chromatography and the target protein was obtained in a polymer free phase with a total recovery of 83% after the two extraction steps.<br/><br>
<br/><br>
The mechanisms for partitioning of plasmid DNA in polymer/polymer systems was determined. A system composed of the thermoseparating EOPO polymer and dextran was developed for purification of plasmid DNA . The plasmid DNA from a desalted lysate could be completely recovered in the EOPO top phase. By increasing the temperature of the top phase, phase separation was induced and the plasmid DNA was obtained in a water phase. The system was effective in concentrating the sample volume with concomitant removal of contaminants e.g. RNA and proteins. The thermoseparating system was also integrated with membrane filtration and restricted access chromatography resulting in a three step process for purification of plasmid DNA. Plasmid DNA was effectively purified from contaminants such as RNA and proteins and was recovered with a total yield of 69% in this process.},
  author       = {Kepka, Cecilia},
  isbn         = {91-628-6222-7},
  keyword      = {Proteins,proteins,plasmid DNA,large-scale,protein purification,integration,Expression,aqueous two-phase systems,enzymology,Proteiner,enzymologi},
  language     = {eng},
  pages        = {143},
  publisher    = {ARRAY(0xbbdc8c8)},
  title        = {Integration of Aqueous Two-Phase Systems into Recovery Processes for Biomolecules},
  year         = {2004},
}