Identification and functional characterisation of enzymes in the glycosylation pathway of Leishmania major [Elektronische Ressource] / von Anne-Christin Lamerz

Lamerz

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2005
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Identification and Functional Characterisation

of Enzymes in the Glycosylation Pathway

of Leishmania major










Dem Fachbereich Chemie
der Universität Hannover
zur Erlangung des Grades

Doktorin der Naturwissenschaften
Dr. rer. nat.

genehmigte Dissertation










von

Dipl.-Biochem. Anne-Christin Lamerz

geboren am 25.12.1974 in Bad Gandersheim


2005




Referentin: Prof. Dr. Rita Gerardy-Schahn
Korreferent: Prof. Dr. Walter Müller
Tag der Promotion: 11. Februar 2005



















































Schlagworte: UDP-Glucose Pyrophosphorylase, UDP-Galactose, Leishmania
Key words: UDP-glucose pyrophosphorylase, UDP-galactose, Leishmania Content i
Abstract………………………………………………………………………………….….1
Zusammenfassung.……………………………………………………………………….3
1 Introduction................................................................................................................. 5
1.1 Leishmania parasites.......................................................................................... 5
1.2 The glycoconjugates of Leishmania................................................................. 7
1.2.1 Structure and biosynthesis of Leishmania glycoconjugates ............................. 7
1.2.2 Implication of the L.major surface glycoconjugates on the survival and
proliferation in the vector and host ............................................................................ 11
1.3 The role of galactose containing conjugates in L.major.............................. 14
1.4 Enzymes involved in the galactose metabolism ........................................... 15
1.4.1 UDP-glucose pyrophosphorylase.................................................................... 15
1.4.2 The UDP-galactose transporter....................................................................... 18
1.5 Glycosylation deficient CHO cells .................................................................. 19
1.6 Aim of this study ............................................................................................... 20
2 Material and Methods…………………………………………………………………...22
2.1 Materials............................................................................................................ 22
2.1.1 Eukaryotic cell lines ................................................................................... 22
2.1.2 Bacterial strains ......................................................................................... 22
2.1.3 Plasmids..................................................................................................... 23
2.1.4 Oligonucleotides ........................................................................................ 28
2.1.5 Chromatography columns.......................................................................... 29
2.1.6 Antibodies .................................................................................................. 30
2.1.7 Molecular weight markers 31
2.1.8 Enzymes 31
2.1.9 Culture media and additives ...................................................................... 31
2.1.10 Kits and further materials........................................................................... 32
2.1.11 Standard buffer and media ........................................................................ 33
2.1.12 Chemicals .................................................................................................. 34
2.1.13 Laboratory Equipment................................................................................ 37
2.2 Cell biological techniques (CHO cells).......................................................... 38
2.2.1 Cultivation of CHO cells ............................................................................. 38
2.2.2 Transient transfection of CHO cells........................................................... 38
2.2.3 Immunocytochemistry 38
2.2.4 Immunofluorescence.................................................................................. 39
2.2.5 FACS analysis of CHO cells ...................................................................... 40
2.2.6 Selection of galactose negative CHO Lec8 cells ...................................... 40
2.3 Cell biological techniques (Leishmania major)............................................ 41 Content ii
2.3.1 Cultivation of Leishmania major (L. major)................................................ 41
2.3.2 Homolgous gene replacment in L. major promastigotes........................... 41
2.3.3 FACS analysis of L. major ......................................................................... 42
2.3.4 Immunofluorescence.................................................................................. 42
2.3.5 Mice infection assay................................................................................... 43
2.4 Biochemical techniques 43
2.4.1 Protein estimation ...................................................................................... 43
2.4.2 Polyacrylamide gelelectrophoresis (SDS-PAGE)...................................... 43
2.4.3 Coomassie staining of polyacrylamide gels .............................................. 44
2.4.4 Silver staining of polyacrylamide gels ....................................................... 44
2.4.5 Western Blotting......................................................................................... 44
2.4.6 Immunostaining of Western Blots.............................................................. 44
2.4.7 Solubilisation of CHO cells ........................................................................ 45
2.4.8 Immunoprecipitations................................................................................. 45
2.4.9 Preparation of L. major lysates .................................................................. 46
2.4.10 Expression of recombinant protein in E.coli .............................................. 46
2.4.11 Purification of UDP-glucose pyrophosphorylase from L. major ................ 47
2.4.12 Size exclusion column ............................................................................... 48
2.4.13 Activity assays of UGP .............................................................................. 49
2.4.14 Production of polyclonal L. major UGP antisera ....................................... 50
2.5 Molecular biological techniques.................................................................... 51
2.5.1 Precipitation of nucleic acids ..................................................................... 51
2.5.2 Phenol Chloroform extraction 52
2.5.3 Hot phenol extraction................................................................................. 52
2.5.4 Determination of DNA and RNA concentrations ....................................... 52
2.5.5 Agarose gel electrophoresis of DNA ......................................................... 53
2.5.6 General cloning techniques ....................................................................... 53
2.5.7 Analytical plasmid preparation................................................................... 54
2.5.8 Preparative plasmid preparation................................................................ 54
2.5.9 Transformation of competent E.coli........................................................... 55
2.5.10 Preparation of chemically competent E.coli .............................................. 55
2.5.11 Preparation of E.coli DMSO stocks 56
2.5.12 Polymerase chain reaction (PCR) ............................................................. 56
2.5.13 Isolation of genomic DNA from Leishmania 57
2.5.14 Southern Blot analysis ............................................................................... 58
2.5.15 Isolation of total RNA and mRNA .............................................................. 59
2.5.16 Agarose gel electrophoresis of RNA ......................................................... 60 Content iii
2.5.17 Generation of a cDNA library..................................................................... 60
2.5.18 Complementation cloning .......................................................................... 61
3 Results……………………………………………………………………………………..62
3.1 Identification of the target gene ...................................................................... 62
3.1.1 Cloning strategy............................................................................................... 62
3.1.2 Generation of a L. major cDNA library ...........