Investigation of the Antimicrobial Properties of Lipid Transport Proteins from Members of the Brassicaceae Family
Translated title
Undersøgelse af de antimikrobielle egenskaber af lipid transport-proteiner fra medlemmer af Brassicaceae familien
Author
Bugge, Katrine
Term
4. term
Education
Publication year
2012
Submitted on
2012-06-13
Pages
139
Abstract
Dette speciale undersøger lipidtransferproteiner (LTP’er) i planter fra korsblomstfamilien (Brassicaceae). LTP’er er små proteiner, der binder og flytter lipider og kan bidrage til planters forsvar. I første del blev to Arabidopsis thaliana-LTP’er, LTP5 og LTP8, sammenlignet. De har meget forskellige isoelektriske punkter (pI 11 og 5), hvilket betyder, at de har meget forskellige ladninger ved fysiologisk pH. 3D-modeller blev bygget ved homologimodellering, og deres overfladers elektrostatiske potentialer blev beregnet og visualiseret. De markante forskelle støtter, at proteiner med ens overordnet fold kan virke på forskellige måder. LTP5 og LTP8 blev derefter forsøgt produceret i gæren Pichia pastoris. Kun små mængder af formodet LTP5 blev påvist i koncentreret kultur-supernatant i ét forsøg, og dette materiale viste antimikrobiel aktivitet mod Bacillus subtilis. Generelt kunne der ikke påvises ophobning af LTP5 eller LTP8 under de testede betingelser, så yderligere karakterisering var ikke mulig inden for projektperioden. I anden del blev der udviklet selektive protokoller til at ekstrahere og isolere formodede LTP’er fra kål. Protokollerne synes velegnede til at isolere LTP’er, men kræver bedre udbytte og gennemløb. Med dem blev tre forskellige medlemmer af LTP-familie 1 med forskellig vævsspecificitet identificeret og delvist isoleret. Lipidtransfer-aktivitetstests blev udført for at bekræfte, at proteinerne er egentlige LTP’er, men resultaterne var inkonklusive. Ved lave koncentrationer viste de formodede LTP-fraktioner ingen antimikrobiel aktivitet mod fire testede mikroorganismer.
This thesis examines lipid transfer proteins (LTPs) in plants of the cabbage/mustard family (Brassicaceae). LTPs are small proteins that bind and move lipids and can contribute to plant defense. In part one, two Arabidopsis thaliana LTPs, LTP5 and LTP8, were compared. They have very different isoelectric points (pI 11 and 5), meaning they carry very different charges at physiological pH. 3D models were built by homology modeling, and their surface electrostatic potentials were calculated and visualized. The marked differences support that proteins with similar overall folds may act in different ways. LTP5 and LTP8 were then expressed in the yeast Pichia pastoris. Only low amounts of putative LTP5 were detected in concentrated culture supernatant in one experiment, and this material showed antimicrobial activity against Bacillus subtilis. Overall, accumulation of LTP5 and LTP8 was not detectable under the conditions tested, so further characterization was not possible within the project period. In part two, selective protocols were developed to extract and isolate putative LTPs from cabbage. The protocols appear suitable for isolating LTPs but need better yield and throughput. Using them, three distinct members of LTP family 1 with different tissue specificities were identified and partially isolated. Lipid transfer activity assays were performed to verify that the proteins are true LTPs, but the results were inconclusive. At low concentrations, the putative LTP fractions showed no antimicrobial activity against four tested microbes.
[This abstract was generated with the help of AI]
Keywords
Documents