Příprava rekombinantních glykosyltransferas z Mycobacterium tuberculosis

Bc. Kristýna Hrnčířová, Ph.D.

Diplomová práce

Anotace:

Tuberkuloza rocne zpsobi na celem svete umrti temer 2 milionu lidi, navic cela tretina lidske populace v sobe v soucasnosti nosi puvodce tohoto onemocneni bakterii Mycobacterium tuberculosis. Lecba tuberkulozy je velmi obtizna, Mycobacterium tuberculosis je totiz zejmena diky specifickemu slozeni sve bunecne steny velmi odolne - vuci obrannym mechanismum lidskeho imunitniho systemu, ale take napr. vuci vstupu antibiotik do bunky. Cela bunecna stena je prostoupena latkami lipidni a voskove povahy, hlavni kostru pak tvori tzv. mykolyl-arabinogalaktan-peptidoglykanovy komplex. Jednou z hlavnich skupin enzymu, ktere se podileji na vystavbe teto bunecne steny, jsou glykosyltransferasy, ktere katalyzuji prenos mono- nebo oligo-sacharidu na akceptor (tim mohou byt sacharidy, proteiny nebo lipidy). Do mykobakterialni bunecne steny tak zabudovavaji specificke sacharidy (napr. rhamnosu) a to navic velmi specifickymi, obtizne stepitelnymi vazbami. Pripadnou inhibici techto enzymu u Mycobacterium tuberculosis by mohl byt ucinen vyrazny posun v lecbe tuberkulozy. Jelikoz je Mycobacterium tuberculosis patogenni organismus, jedinou bezpecnou moznosti studia jeho proteinu je tzv. rekombinantni metoda, pri niz je gen pro protein vnesen do vhodneho vektoru a exprimovan v hostitelskych bunkach. Glykosyltransferasy, ktere jsou zpravidla membranove, nebo alespon asociovane s membranami, a tedy hydrofobni, jsou vsak v hostitelskych organismech jen velmi obtizne exprimovatelne. Pokud k expresi vubec dochazi, byva velka cast exprimovana do neaktivnich inkluznich telisek. Tato diplomova prce byla zamerena na expresi a purifikaci rekombinantnich glykosyltransferas Mycobacterium tuberculosis za pouziti E. coli jako hostitelskeho organismu. Ziskanim cistych glykosyltransferas by byla mozna jejich strukturni a funkcni charakterizace.

Abstract:

2 milion people worldwide die every year due to tuberculosis and one third of world population is infected with its causative agent, pathogenic bacteria Mycobacterium tuberculosis. Treatment of tuberculosis is very difficult, mainly because of very specific constitution of mycobacterial cell wall that defends mycobacteria against damage or entry of antibiotics. The core of the cell wall consists of so called mycolyl-arabinogalactan-peptidoglycan complex and is surrounded with several lipids and waxes. Lipoarabinomannan passes through whole cell wall. The main group of enzymes which participate in synthesis of mycobacterial cell wall are glycosyltransferases that catalyse the transfer of an activated saccharide unit to an acceptor (that can be sacharides, lipids or proteins). By inhibition of these enzymes we could disrupt the cell wall synthesis for treatment of tuberculosis. M. tuberculosis belongs to pathogens, so the only way to study mycobacterial proteins is in recombinant form. It means to clone the gene encoding the protein into a vector and to express the protein in a host cells (for example in E. coli). Expression of glycosyltransferases in host organisms is very problematic because of their hydrophobicity and toxicity. To optimize preparation of soluble and pure protein different expression systems have been tested. The thesis was focused on three glycosyltransferases from M. tuberculosis: Rv1526c, Rv2962c and Rv3782. There were applied different approaches to obtain soluble proteins. Three fusion MBP(maltose binding protein)-glycosyltransferase proteins were succesfully prepared. But cleavage of MBP from fusion proteins didnt acquire pure glycosyltransferase. Rv3782 fused with His-tag was succesfully refolded (although with low purity) on column from inclusion bodies. There have been started work on a new vectors pMAL-TWIN-L1 and pMAL-TWIN-L3 which should join the good properties of two commercial vectors pMAL-c2E (MBP helps with folding of hydrophobic proteins) and pTWIN1 (inteins could help with cleavage of MBP from fusion protein). One new vector (pMAL-TWIN-L1) was prepared and is ready to be tested in further cloning.

Klíčová slova

glykosyltransferasy, glykosylace, Mycobacterium tuberculosis, tuberkuloza, rekombinantni proteiny

Jazyk práce: čeština

Datum vytvoření / odevzdání či podání práce: 28. 4. 2006

Obhajoba závěrečné práce

• Obhajoba proběhla 23. 5. 2006
• Vedoucí: prof. RNDr. Michaela Wimmerová, Ph.D.

Masarykova univerzita

Přírodovědecká fakulta

Magisterský studijní program / obor:
Biochemie / Biochemie