Development of universal transmembrane transporter systems [Elektronische Ressource] / presented by Markus Andreas Moosmeier

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DISSERTATION submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola-University Heidelberg, Germany, for the degree of Doctor of Natural Sciences presented by Markus Andreas Moosmeier, M.Sc. born in Landshut, Germany Date of oral examination: 2009/07/23 DEVELOPMENT OF UNIVERSAL TRANSMEMBRANE TRANSPORTER SYSTEMS Referees: Professor Dr. Stephan Urban Professor Dr. Hanswalter Zentgraf To my parents and Andrea, for their endless love and encouragement. “In the beginning, I looked around and could not find quite the protein I dreamed of. So I decided to build it myself.” Freely adapted from Dr. Ing. h.c. Ferdinand Porsche Table of Contents SUMMARY ................................................................................................................................................ 1 PUBLICATIONS AND PATENTS.............................................................................................................. 9 CHAPTER 1: INTRODUCTION............................................................................................................... 13 1.1 Targeted Therapy ......................................................................................................................... 13 1.2 Transmembrane Delivery of Therapeutic Agents .........................................

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Published 01 January 2009
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DISSERTATION
submitted to the
Combined Faculties for the Natural Sciences
and for Mathematics
of the Ruperto-Carola-University Heidelberg, Germany,
for the degree of
Doctor of Natural Sciences








presented by
Markus Andreas Moosmeier, M.Sc.
born in Landshut, Germany
Date of oral examination: 2009/07/23


DEVELOPMENT OF
UNIVERSAL TRANSMEMBRANE
TRANSPORTER SYSTEMS




Referees: Professor Dr. Stephan Urban
Professor Dr. Hanswalter Zentgraf












To my parents and Andrea,
for their endless love and encouragement.



















“In the beginning, I looked around
and could not find quite the protein I dreamed of.
So I decided to build it myself.”
Freely adapted from Dr. Ing. h.c. Ferdinand Porsche

Table of Contents
SUMMARY ................................................................................................................................................ 1
PUBLICATIONS AND PATENTS.............................................................................................................. 9
CHAPTER 1: INTRODUCTION............................................................................................................... 13
1.1 Targeted Therapy ......................................................................................................................... 13
1.2 Transmembrane Delivery of Therapeutic Agents ......................................................................... 15
1.3 Universal Transmembrane Delivery Systems............................................................................... 18
1.4 Research Objectives............. 21
CHAPTER 2: RESULTS............................................................................................................. 25
2.1 Construction, Expression, and Purification of Streptavidin and its Derivatives............................. 25
2.2 Biophysical Characterization of Streptavidin and its Derivatives .................................................. 28
2.3 Internalization of Streptavidin and Strep-Tactin by PTD-Fused Ligands ...................................... 37
2.4 Internalization of Transvidins and Transtactins.............................................................................46
2.5 Transvidins as Universal Delivery Systems for Biotinylated Cargos............................................. 51
2.6 Transtactins as Universal Delivery Systems for Strep-tag II-Fused Cargos................................. 55
CHAPTER 3: DISCUSSION.................................................................................................................... 61
3.1 Transmembrane Delivery of Cargos............................................................................................. 61
3.2 Internalization of Non-Modified Cargos......................................................................................... 61
3.3 Transvidin and Transtactin Transporters ...................................................................................... 63
3.4 Therapeutic Implications............................................................................................................... 69
CHAPTER 4: MATERIALS AND METHODS .......................................................................................... 73
4.1 Materials ....................................................................................................................................... 73
4.2 Molecular Biology Methods........................................................................................................... 78
4.3 Biophysical Methods..................................................................................................................... 85
4.4 Cytology Methods ......................................................................................................................... 87
ABBREVIATIONS AND SYMBOLS......................................................................................................... 95
REFERENCES...................................................................................................................................... 101