Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle:
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis/Dissertation Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Links: | http://edoc.ub.uni-muenchen.de/14947/ http://d-nb.info/1027669441/34 https://nbn-resolving.org/urn:nbn:de:bvb:19-149476 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025668281&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| Physical Description: | 162 Bl. Ill., graph. Darst. |
Staff View
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| 245 | 1 | 0 | |a Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle |c Kevin Maier |
| 264 | 1 | |c 2012 | |
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Record in the Search Index
| DE-BY-TUM_katkey | 1906880 |
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| adam_text | IMAGE 1
TABLE OF CONTENTS 5
1 TABLE OF CONTENTS
1 TABLE OF CONTENTS 5
2 INTRODUCTION 11
2.1 MACROMOLECULAR THERAPY - A BRIEF SURVEY 11
2.2 BARRIERS IN MACROMOLECULAR THERAPY 13
2.3 NUCLEIC ACID VERSUS PROTEIN DELIVERY 17
2.4 STRATEGIES FOR TRANSPORTING PROTEINS INTO THE CYTOSOL OF LIVING
CELLS 19
2.5 BIOLOGICAL CLEAVEABLE CROSSLINKERS 21
2.6 AIMS OF THE THESIS 23
2.6.1 ESTABLISHMENT OF A CELLULAR TEST SYSTEM FOR PROTEIN DELIVERY 23
2.6.2 PROTEIN DELIVERY WITH DEFINED POLYCATIONS 24
2.6.3 DEVELOPMENT OF A NEW TRACELESS CLEAVABLE LINKER FOR PROTEIN
DELIVERY... 25
3 MATERIALS 27
3.1 CHEMICALS 27
3.2 REAGENTS 27
3.2.1 DYES 27
3.2.2 PEPTIDES 27
3.2.3 LINKERS 27
3.3 SOLVENTS 28
3.4 BUFFERS 28
3.5 SOLUTIONS 28
3.6 CELL CULTURE MATERIAL 28
HTTP://D-NB.INFO/1028385161
IMAGE 2
TABLE OF CONTENTS 6
3.7 CELL LINES 29
3.8 BACTERIA STRAINS 29
3.9 NUCLEIC ACIDS 29
4 METHODS 30
4.1 GENERAL CHEMICAL PROCEDURES 30
4.1.1 1 HNMRAND 13 CNMR 30
4.1.2 MASS SPECTROSCOPY 30
4.1.3 CD SPECTROSCOPY 30
4.1.4 UV-VIS SPECTROSCOPY 31
4.1.5 FLUORESCENCE SPECTROSCOPY 31
4.1.6 CHROMATOGRAHY 31
4.1.7 DYNAMIC LIGHT SCATTERING (DLS) 32
4.2 SYNTHESIS 32
4.2.1 SYNTHESIS OF WATERSOLUBLE HEMICYANINE ALKYNE DYE 32
4.2.2 SYNTHESIS OF AZIDE HEMICYANINE DYE 34
4.2.3 SYNTHESIS OF N-PROPARGYL MALEIMIDE 36
4.2.4 SYNTHESIS OF N-SUCCININIMIDYL-3-(2-PYRIDYLDITHIO)-PROPIONATE
(SPDP)... 36
4.2.5 SYNTHESIS OF MAM LINKER 38
4.2.6 SYNTHESIS OF AZMMMAN LINKER 39
4.2.7 SYNTHESIS OF PENTYNEMMAN LINKER 40
4.3 CLONING, HETEROLOGOUS EXPRESSION AND PURIFICATION OF NLSEGFP AND
HIV-TATNLSEGFP 42
4.4 MODIFICATION OF PROTEINS WITH CROSSLINKERS 43
4.4.1 MODIFICATION WITH SPDP 43
4.4.2 MODIFICATION OF NLSEGFP WITH SUCCINIMIDYL-4-(/V-MALEIMIDOMETHYL) R
CYCLOHEXANE-1-CARBOXYLATE (SMCC) 44
4.4.3 MODIFICATION OF NLSEGFP WITH MAM LINKER 44
IMAGE 3
TABLE OF CONTENTS 7
4.4.4 MODIFICATION OF HSA WITH PENTYNEMMAN LINKER 44
4.4.5 MODIFICATION WITH AZMMMAN LINKER 45
4.5 COUPLING OF CARRIERS, DYES AND PEG TO THE PROTEINS 46
4.5.1 COUPLING OF G3 PPI TO NLSEGFP VIA EDC 46
4.5.2 COUPLING OF G3 PPI TO NLSEGFPX5SPDP VIA A REDUCIBLE DISULFIDE BOND
47
4.5.3 COUPLING OF 386 TO NLSEGFPX5SPDP 47
4.5.4 COUPLING OF 386 TO B-GALACTOSIDASEX8SPDP 48
4.5.5 COUPLING OF 386 TO SMCC-NLSEGFP 48
4.5.6 COUPLING OF 386 TO AZMMMAN-NLSEGFP 48
4.5.7 COUPLING OF TMR LABELLED-386 TO AZMMMAN-NLSEGFP 48
4.5.8 COUPLING OF 386 TO AZMMMAN-IJ-GALACTOSIDASE 49
4.5.9 COUPLING OF POLY(ETHYLENE GLYCOL) (PEG) 49
4.5.10 COUPLING OF ALKYNE HEMICYANINE DYE TO AZMMMAN-HSA 50
4.5.11 COUPLING OF PHOSPHINE DYE DYLIGHT 488 TO AZMMMAN-HSA 50
4.5.12 COUPLING OF AZIDE DYE TO PENTYNEMMAN-HSA 51
4.5.13 COUPLING OF FOLIC ACID AND MELITTIN TO AZMMMAN-EGFP 51
4.5.14 COUPLING OF TRANSDUCTION OLIGOMER 71 TO MAM-EGFP 52
4.6 CHARACTERIZATION OF NEW ACID LABILE CROSSLINKERS 52
4.6.1 DETERMINATION OF THE DEGREE OF PROTEIN MODIFICATION 52
4.6.2 DETERMINATION OF THE ACID CATALYZED RELEASE OF LINKER FROM THE
PROTEIN.. 53
4.6.3 SERUM STABILITY ASSAY 53
4.6.4 RELEASE OF PEG FROM HSA CONJUGATE 54
4.6.5 RELEASE KINETICS OF TMR-LABELED PEG FROM HSA CONJUGATE 54
4.6.6 ACID CATALYZED RELEASE OF NLSEGFP FROM 386-DBCO -AZMMMANNLSEGFP 55
4.7 BIOLOGICAL CHARACTERIZATION OF THE SYNTHESIZED CONJUGATES 56
4.7.1 GENERAL PROCEDURES 56
IMAGE 4
TABLE OF CONTENTS 8
4.7.2 PROTEIN TRANSDUCTIONS 57
4.7.3 ENDOCYTOSIS INHIBITION 59
4.7.4 CELL VIABILITY ASSAY 59
4.7.5 ACTIVITY TEST OF MODIFIED B-GALACTOSIDASE 59
4.7.6 ERYTHROCYTE LEAKAGE ASSAY 60
5 RESULTS 61
5.1 NLSEGFP AS A MODEL PROTEIN 61
5.1.1 CLONING, HETEROLOG EXPRESSION AND PURIFICATION OF NLSEGFP AND
TATNLSEGFP 62
5.1.2 MICROINJECTION OF NLSEGFP AND TAT-NLSEGFP 64
5.2 PROTEIN TRANSDUCTION WITH TAT-NLSEGFP 66
5.3 CATIONIC LIPID MEDIATED PROTEIN DELIVERY 68
5.4 DENDRIMER BASED PROTEIN TRANSDUCTION 68
5.4.1 SYNTHESIS OF G3 PPI-NLSEGFP 68
5.4.2 PROTEIN TRANSDUCTION WITH G3 PPI-NLSEGFP 70
5.4.3 MICROINJECTION OF G3 PPI-NLSEGFP 72
5.5 PROTEIN TRANSDUCTION WITH G3PPI COUPLED TO NLSEGFP OVER A
BIOREVERSIBLE
BOND 73
5.5.1 SYNTHESIS OF G3PPI-SS-NLSEGFP 73
5.5.2 PROTEIN TRANSDUCTION USING G3PPI-SS-NLSEGFP 75
5.6 PROTEIN TRANSDUCTION USING THE STRUCTURE DEFINED OLIGOMER 386 77
5.6.1 SYNTHESIS OF REDUCIBLE 386 MODIFIED NLSEGFP 77
5.6.2 PROTEIN TRANSDUCTION WITH 386 -SS-NLSEGFP 79
5.6.3 TRANSDUCTION OF 386 -SS-IJGALACTOSIDASE 85
5.7 DEVELOPMENT OF ACID LABILE, TRACELESS HETEROBIFUNCTIONAL CLICK
LINKERS FOR PROTEIN
DELIVERY 88
5.7.1 SYNTHESIS OF MAM LINKER 88
IMAGE 5
TABLE OF CONTENTS 9
5.7.2 ACID LABILITY OF MAM LINKER 89
5.7.3 SYNTHESIS OF AZMMMAN LINKER 91
5.7.4 SYNTHESIS OF WATER SOLUBLE ALKYNE HEMICYANINE DYE 92
5.7.5 MODIFICATION OF PROTEINS WITH AZMMMAN 93
5.7.6 SYNTHESIS OF PENTYNEMMAN CROSSLINKER 106
5.7.7 ACID LABILITY OF PENTYNEMMAN LINKER 107
6 DISCUSSION 109
6.1 ESTABLISHING OF A CELLULAR TEST SYSTEM FOR PROTEIN DELIVERY 109
6.2 DEVELOPMENT OF A SEQUENCE DEFINED CARRIER SYSTEM FOR PROTEIN
DELIVERY ... 110
6.3 DESIGN OF A TRACELESS-CLEAVABLE LINKER FOR PROTEIN DELIVERY 114
7 SUMMARY 117
8 ABBREVIATIONS 119
9 APPENDIX 124
9.1 PLASMID MAPS AND BASE SEQUENCES 124
9.1.1 PEL 068-PRHGPCNA 124
9.1.2 PET 23A(+) 128
9.1.3 PET 23A(+)-SV40NLS-EGFP 131
9.1.4 PET 23A(+)-SV40NIS-EGFP-HIVTAT 134
9.2 AMINO ACID SEQUENCES 137
9.2.1 SV40NLS-EGFP 137
9.2.2 SV40NLS-EGFP-H IVTAT 137
9.2.3 R-GALACTOSIDASE 137
9.3 ANALYTIC SPECTRA 139
9.3.1 ALKYNE HEMICYANINE DYE 139
9.3.2 AZMMMAN LINKER 140
9.3.3 PENTYNEMMAN LINKER 143
10 REFERENCES 147
IMAGE 6
TABLE OF CONTENTS 10
11 PUBLICATIONS 159
11.1 ORIGINAL PAPERS 159
11.2 BOOK CHAPTERS 159
11.3 POSTER PRESENTATIONS 160
12 ACKNOWLEDGEMENTS 161
|
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| indexdate | 2024-12-20T16:23:22Z |
| institution | BVB |
| language | English |
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| physical | 162 Bl. Ill., graph. Darst. |
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| spellingShingle | Maier, Kevin Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle |
| subject_GND | (DE-588)4113937-9 |
| title | Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle |
| title_auth | Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle |
| title_exact_search | Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle |
| title_full | Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle Kevin Maier |
| title_fullStr | Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle Kevin Maier |
| title_full_unstemmed | Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle Kevin Maier |
| title_short | Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle |
| title_sort | design of an acid labile traceless cleavable click linker for use in a novel protein transduction shuttle |
| topic_facet | Hochschulschrift |
| url | http://edoc.ub.uni-muenchen.de/14947/ http://d-nb.info/1027669441/34 https://nbn-resolving.org/urn:nbn:de:bvb:19-149476 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025668281&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT maierkevin designofanacidlabiletracelesscleavableclicklinkerforuseinanovelproteintransductionshuttle |