Advanced

HYDROPHOBIC PEPTIDE TAGS - As tools in bioseparation and investigation of recombinant proteins.

Fexby, Sara LU (2003)
Abstract (Swedish)
Popular Abstract in Swedish

Många framsteg har gjorts inom bioteknik de senaste decennierna. Idag kan vissa sjukdomar fastställas redan på DNA-nivå och viktiga läkemedel samt tvättmedelskomponenter tillverkas industriellt med hjälp av gentekniska tekniker. Kostnaden för de gentekniska produkterna ligger till stor del i själva uppreningsprocessen. Forskningen i denna avhandling har gjorts med avsikt att utveckla nya metoder samt förbättra redan befintliga tekniker för att rena upp gentekniska proteiner.



I genteknik kan man använda sig av bakterier för att producera proteiner. En bakterie är en levande organism som själv ombesörjer allt, från värmeförsörjning till rörelse, bara den får näring. Man skulle... (More)
Popular Abstract in Swedish

Många framsteg har gjorts inom bioteknik de senaste decennierna. Idag kan vissa sjukdomar fastställas redan på DNA-nivå och viktiga läkemedel samt tvättmedelskomponenter tillverkas industriellt med hjälp av gentekniska tekniker. Kostnaden för de gentekniska produkterna ligger till stor del i själva uppreningsprocessen. Forskningen i denna avhandling har gjorts med avsikt att utveckla nya metoder samt förbättra redan befintliga tekniker för att rena upp gentekniska proteiner.



I genteknik kan man använda sig av bakterier för att producera proteiner. En bakterie är en levande organism som själv ombesörjer allt, från värmeförsörjning till rörelse, bara den får näring. Man skulle kunna jämföra en bakterie med en fabrik. Här sköts allt från kundordern (genomet, d.v.s. DNA:t) till färdig produkt (protein), bara energi tillförs så att maskineriet fungerar. Med gentekniska tekniker kan man förändra bakteriens DNA och på så sätt tvinga bakterien att producera ett protein som normalt inte existerar i bakterien. I en vanlig bakterie finns över 2000 olika proteiner, som bakterien behöver för att kunna fungera. För att kunna urskilja och hitta vårt önskade protein kan vi märka det. Protein kan märkas så att de syns i en blandning, på samma sätt som en turistguide kan bära ett paraply för att synas i en större folkmängd.



I min forskning har jag gentekniskt märkt proteiner med små handtag som ger proteinet unika egenskaper. Dessa egenskaper kan utnyttjas för att ”fiska upp” det önskade proteinet ur en proteinblandning. Svårigheten är att designa handtagen så finurligt som möjligt att målproteinet effektivt kan avskiljas från föroreningar. Handtagen som fästs på målproteinet får inte skada eller förstöra målproteinets naturliga egenskaper.



Lyckades jag? Visst har jag lyckats designa flera olika handtag (hydrophobic peptide tags) och gentekniskt satt dem på mina önskade proteiner (lactate dehydrogenase, LDH, och green fluorescent protein, GFPuv). Mina proteiner har då fått speciella egenskaper och dessa har undersökts och utnyttjas för upprening av proteiner, vilket beskrivs i avhandlingen. Förutom design av handtag har ett nytt ”fiskespö” utvecklats, det vill säga den fasta yta som fångar upp proteinerna (HBVE/BVE medium). (Less)
Abstract
This thesis describes the possibility of using hydrophobic peptide tags as tools for bioseparation and for the investigation of recombinant proteins. The hydrophobicity of a protein can easily be changed by minor modifications, such as fusions of tyrosine-containing peptides. The fusion tags may also increase both stability and expression of the fusion protein.



Furthermore, a protein library with randomised pentapeptides was created, and the variants exhibited significant differences in relative hydrophobicity, although the pentapeptides only caused minor alterations in primary structure, size and isoelectric point of the fusion proteins. The changes in relative hydrophobicity were measured with aqueous two-phase systems... (More)
This thesis describes the possibility of using hydrophobic peptide tags as tools for bioseparation and for the investigation of recombinant proteins. The hydrophobicity of a protein can easily be changed by minor modifications, such as fusions of tyrosine-containing peptides. The fusion tags may also increase both stability and expression of the fusion protein.



Furthermore, a protein library with randomised pentapeptides was created, and the variants exhibited significant differences in relative hydrophobicity, although the pentapeptides only caused minor alterations in primary structure, size and isoelectric point of the fusion proteins. The changes in relative hydrophobicity were measured with aqueous two-phase systems (ATPSs) and hydrophobic interaction chromatography (HIC), and the results indicated the sensitivity of both methods to slight variations in the primary structure of proteins and their surface hydrophobicity. The results of the techniques correlated well, and the correlation coefficient, R2, was 0.89.



Upon comparing the relative hydrophobicity contributions from seventeen LDH variants from thirty-four hydrophobicity scales they were found to correlate fairly well with experimental results from ATPSs and HIC. However, the results from such scales should only be used as a complement to experimental studies, for example, to indicate tag exposure.



A novel HIC adsorbent, HBVE/BVE, was developed with polymer grafting techniques. The advantage of the HBVE/BVE medium is the possibility to alter the hydrophobic properties of the medium by only changing the reaction ratio of the two monomers in the grafting procedure. The hydrophobicity can be tuned to improve protein purification. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Prof Queiroz, Joãn, Faculty of Health Sciences, University of Beira Interior, Covilha, Portugal.
organization
publishing date
type
Thesis
publication status
published
subject
keywords
E. coli, Biochemical technology, Biokemisk teknik, fusion protein, tyrosine, hydrophobicity, Hydrophobic interaction chromatography, aqueous two-phase system
pages
105 pages
publisher
Department of Pure and Applied Biochemistry, Lund University
defense location
Center for Chemistry and Chemical Engineering, Lecture Hall B, Lund Institute of Technology.
defense date
2003-11-27 10:30
ISBN
91-7422-037-3
language
English
LU publication?
yes
id
92822ddc-42d2-43d4-a925-df7f10ae9c0b (old id 466464)
date added to LUP
2007-10-14 13:38:08
date last changed
2016-09-19 08:45:15
@misc{92822ddc-42d2-43d4-a925-df7f10ae9c0b,
  abstract     = {This thesis describes the possibility of using hydrophobic peptide tags as tools for bioseparation and for the investigation of recombinant proteins. The hydrophobicity of a protein can easily be changed by minor modifications, such as fusions of tyrosine-containing peptides. The fusion tags may also increase both stability and expression of the fusion protein.<br/><br>
<br/><br>
Furthermore, a protein library with randomised pentapeptides was created, and the variants exhibited significant differences in relative hydrophobicity, although the pentapeptides only caused minor alterations in primary structure, size and isoelectric point of the fusion proteins. The changes in relative hydrophobicity were measured with aqueous two-phase systems (ATPSs) and hydrophobic interaction chromatography (HIC), and the results indicated the sensitivity of both methods to slight variations in the primary structure of proteins and their surface hydrophobicity. The results of the techniques correlated well, and the correlation coefficient, R2, was 0.89.<br/><br>
<br/><br>
Upon comparing the relative hydrophobicity contributions from seventeen LDH variants from thirty-four hydrophobicity scales they were found to correlate fairly well with experimental results from ATPSs and HIC. However, the results from such scales should only be used as a complement to experimental studies, for example, to indicate tag exposure.<br/><br>
<br/><br>
A novel HIC adsorbent, HBVE/BVE, was developed with polymer grafting techniques. The advantage of the HBVE/BVE medium is the possibility to alter the hydrophobic properties of the medium by only changing the reaction ratio of the two monomers in the grafting procedure. The hydrophobicity can be tuned to improve protein purification.},
  author       = {Fexby, Sara},
  isbn         = {91-7422-037-3},
  keyword      = {E. coli,Biochemical technology,Biokemisk teknik,fusion protein,tyrosine,hydrophobicity,Hydrophobic interaction chromatography,aqueous two-phase system},
  language     = {eng},
  pages        = {105},
  publisher    = {ARRAY(0xc23a0f8)},
  title        = {HYDROPHOBIC PEPTIDE TAGS - As tools in bioseparation and investigation of recombinant proteins.},
  year         = {2003},
}