Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Production of gangliosidebiosynthetic membrane enzymes for biochemical and functionalstudies: Expression, purification and crystallizationoptimization of Thermococcus onnurineus Dolicho l-phosphate mannose synthase, Homosapiens and Branchiostoma floridae Glucosylceramide synthase
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
2018 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Glycolipids play important roles in the biology of prokaryotes and eukaryotes, including humans, and although theyare found on the cell-membrane surface of all eukaryotic cells, not much is known about their biosynthesis. The aim ofthis project was to characterize two enzymes: glucosylceramide synthase (GCS) which is involved in the biosynthesisof glycolipids such as gangliosides that are abundant in the membranes of nerve cells; and dolicholphosphate mannosesynthase (DPMS), involved in the synthesis precursor for protein glycosylation. Both GCS and DPMS have beenshown play a role in cancer as well as in congenital disorders of glycosylation, and are therefore interesting targets tostudy from a therapeutic perspective.With the goal to identify a suitable expression system for GCS, the genes coding for GCS from lancelet(Branchiostoma floridae) and human (Homo sapiens) were cloned and tested for expression in Escherichia coliBL21(DE3)T1 and C41(DE3) using different vectors. Cloning into three different vectors was successful and initialexpression testing was performed. SDS-PAGE analysis confirmed initial expression of proteins. Although the correctsize of the protein could be confirmed by Western blot, no fluorescence of the GFP-fusion protein could be detected.DPMS from Thermococcus onnurineus (ToDP) was expressed in E. coli C41(DE3) and purified by immobilizedmetal ion affinity chromatography and gel filtration. Crystallization optimization was performed for ToDP producedfrom the vector pNIC28-Bsa4 and plate-like crystals were obtained. X-ray intensity data analysis indicated that thesecrystals contained lipid rather than protein. Crystallization screening for ToDP produced from the vector pNIC-CTHOconstruct was successful. Crystallization screening using the commercially available MemGold-HT96 crystallizationkit resulted in initial crystallization that yielded protein crystals that diffracted to 10 °A resolution.

Abstract [en]

Glykolipider är viktiga biologiska byggstenar hos prokaryoter och eukaryoter, även människor. Trots attglykolipider finns på cellmembranytan hos alla eukaryota celler är inte mycket känt kring syntesen av glykolipider. Målet med detta projekt var att karaktärisera två enzym: glukosylceramidsyntas (GCS) som är involverat i biosyntesenav glykolipider som gangliosider vilka förekommer i cellmembranet hos människors nervceller; och dolikolfosfatmannossyntas (DPMS) som är involverat i syntesen av substrat för proteinglykosylering. Både GCS och DPMS harvisat sig spela en roll i cancer och medfödda glykosyleringssjukdomar och är därför intressanta enzym att studera urett medicinskt perspektiv.Med målet att identifiera ett lämpligt expressionssystem för GCS, klonades gener från lansett (Branschiostomafloridae) och människa (Homo sapiens) och testades för expression i Escherichia coli BL21(DE3)T1 och C41(DE3)med olika vektorer. Kloning av tre olika vektorer lyckades och expressionstester utfördes. Analys med SDS-PAGEbekräftade expression av protein. Trots att korrekt storlek av proteinet kunde bekräftas medWestern blot, detekteradesingen fluorescens från GFP-fusionsproteinet. DPMS från Thermococcus onnurineus (ToDP) i två olika konstrukt uttrycktes i E. coli C41(DE3) och renades med immobiliserad metalljonaffinitetskromatografi och gelfiltrering. Kristalliseringsoptimering utfördes för ToDP uttryckt i vektorn pNIC28-Bsa4 och skivliknande kristaller erhölls. Diffraktionsdata indikerade dockatt kristallerna innehöll lipider och inte protein. Kristallisering av ToDP uttryckt i vektorn pNIC-CTHO lyckadesoch initiala kristallingsförhållanden hittades genom att använda det kommersiellt tillgängliga kristalliseringskitet MemGold-HT96. Diffraktionsdata visade på upplösning ner till 10 Å.

Place, publisher, year, edition, pages
2018.
Keywords [en]
glycoconjugates, glycolipids, integral membrane proteins, gangliosides, glucosylceramide synthase, dolicholphosphate mannose synthase, cancer, neuroblastoma, congenital disorders of glycosylation
Keywords [sv]
glykokonjukat, glykolipider, membranprotein, gangliosider, glukosylceramidsyntas, dolicholfosfat mannossyntas, cancer, neuroblastom, medfdd glykosyleringssjukdom
National Category
Other Engineering and Technologies
Identifiers
URN: urn:nbn:se:kth:diva-230765OAI: oai:DiVA.org:kth-230765DiVA, id: diva2:1219274
Available from: 2018-06-21 Created: 2018-06-15 Last updated: 2018-06-21Bibliographically approved

Open Access in DiVA

fulltext(267 kB)17 downloads
File information
File name FULLTEXT01.pdfFile size 267 kBChecksum SHA-512
4ac6cf9b7dccef1110c3afdff76260ff67837e6b510e012dd1c069a3880edd09b76e306b12c5fa98089f56fd7a21eaee17d2578d0ed47faac85c7f1756f63bcb
Type fulltextMimetype application/pdf

By organisation
School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH)
Other Engineering and Technologies

Search outside of DiVA

GoogleGoogle Scholar
Total: 17 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

urn-nbn

Altmetric score

urn-nbn
Total: 44 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf