Construction of an efficient secretion system for recombinant proteins in Bacillus subtilis [Elektronische Ressource] / Kelly Cristina Leite. Betreuer: Wolfgang Schumann

Wolfgang Schumann

Sujets

• Biologie

Informations

• Publié par: universitat_bayreuth
• Publié le: 01 janvier 2011
• Nombre de lectures: 32
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Construction of an efficient secretion
system for recombinant proteins in
Bacillus subtilis



Dissertation
Zur Erlangung des Grades eines
Doktors der Naturwissenschaften



-Dr. Rer. Nat.-
Der Fakultät für Biologie, Chemie und
Geowissenschaften der Universität Bayreuth



Vorgelegt von
Kelly Cristina Leite
aus
Brasilien




Bayreuth, 2011























To my parents







Die vorliegende Arbeit wurde in der Zeit von April 2008 bis April 2011 am Lehrstuhl für
Genetik der Universität Bayreuth unter der Betreuung von Prof. Dr. Wolfgang Schumann
angefertig.


Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und Geowissenchaften der
Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften (Dr. rer. nat.)


Amtierender Dekan: Prof. Dr. Stephan Clemens
Tag des Einreichens der Dissertation: 30. März 2011
Tag des wissenschaftlichen Kolloquiums: 20. Juni 2011


Prüfungsausschuß:
Prof. Dr. Wolfgang Schumann (Erstgutachter)
Prof. Dr. Harold Drake (Zweitgutachter)
PD Dr. Stefan Heidmann (Vorsitzender)
Prof. Dr. Wulf Blankenfeldt
Prof. Dr. Franz Meußdoerffer










Acknowledgments

I would like to express my singular gratefulness to Professor Wolfgang Schumann, who
unconditionally guided and supported this study. I am really happy for having had the
opportunity to work with him and to learn from his enormous wealth of scientific knowledge.
I am also forever thankful for his kindness and help throughout my stay in Bayreuth.

I appreciate the excellent assistance from Dr. Thomas Wiegert and Dr. Markus Helfrich for
their innumerous occasions for advice and helpful discussions in the course of this work.

I would like to show immense gratitude to all the members in Professor Schumann‟s
laboratory, especially to Katharina Schäfer, Quynh Anh Nguyen and Bach Hue Nguyen; and
in the Biology Department, all of whom are too numerous to list here, for their help with
research insights and wonderful friendships. And a special mention of Joana Bandola, for the
research assistance in the construction of this work.

I am thankful to Karin Angermann and Petra Helies for their valuable assistance and kindness
in making our laboratory a great place to work.

A special thanks to my friends Octavio Flores, Anais Graterol, Johannes Martini and Elisa
Guimaraes for their inestimable friendship; always sharing great moments and for their
constant support and encouragement.

I would like to thank Bayerischeforschungsstifung for its financial support.

I am eternally and extremely thankful to my family for their love and care which gives me the
greatest motivation in life and for their immeasurable enthusiasm and encouragement with
every step I take.

And last but not least, I am thankful for the best thing that has ever happened in my life, my
fiancé Derrick Mulder, who has been by my side through distance and time, always helping
me in any possible way with his love and patience.






Table of Contents

Summary .................................................................................................................................. IV

Zusammenfassung .................................................................................................................... VI

1 Introduction ......................... 1
1.1 Protein traffic: The key role of signal peptides ........................................................... 1
1.2 Secretion of proteins: The pathways ........................................................................... 5
1.2.1 The post-translational translocation mechanism: The Sec pathway and the role
of SecB and SecA in E. coli ................................................................................................ 5
1.2.2 The protein-conducting channel: SecYEG ........................... 7
1.2.3 The Sec pathway: The mechanism of post-translational translocation ................ 9
1.2.4 The co-translational translocation mechanism: The role of SRP and its receptor .
11
1.2.5 The YidC pathway ............................................................................................. 15
1.3 The organism: B. subtilis ........................... 16
1.4 Objectives of the PhD thesis ...................... 21

2 Materials and methods ...................................................................................................... 22
2.1 Bacterial strains, plasmids, oligonucleotides, antibiotics, antibodies and media ...... 22
2.1.1 Bacterial strains and plasmids ............ 22
2.1.2 Oligonucleotides ................................................................................................. 25
2.1.3 Antibiotics .......... 28
2.1.4 Antibodies .......................................................................................................... 28
2.1.5 Media .................. 29
2.2 Enzymes, biochemicals, chemicals and kits .............................................................. 29
2.2.1 Enzymes ............................................................................................................. 29
2.2.2 Biochemicals and chemicals .............. 29
2.2.3 Kits ..................................................................................................................... 30
2.3 General methods ........ 30
2.3.1 PCR .................... 30
2.3.2 Cloning ............................................................................................................... 31


I



2.3.3 Growth and collection of samples ...................................................................... 31
2.4 RNA: Northern blot analysis ..................................................................................... 32
2.4.1 Isolation of total RNA from B. subtilis .............................. 32
2.4.2 Electrophoresis of RNA and vacuum blot transfer to membranes ..................... 32
2.4.3 Transcriptional labeling of RNA probes ............................................................ 32
2.4.4 Hybridization of membrane-bound RNA with RNA probes ............................. 33
2.4.5 Stripping of RNA probes .................................................................................... 33
2.5 Protein: Western blot analysis ................... 33
2.5.1 Preparation of soluble and insoluble cell extracts from B. subtilis .................... 33
2.5.2 Determination of protein concentration ............................................................. 33
2.5.3 Precipitation of proteins from culture supernatants ........................................... 34
2.5.4 Western blot analysis ......................................................... 34
2.6 Visualization and measurement of reporter gene expression .... 34
2.6.1 Visualization of extracellular enzyme activity of α-amylase on plates .............. 35
2.6.2 Measurement of the α-amylase activity ............................................................. 35
2.6.3 Measurement of the β-galactosidase activity ..................... 35
2.6.4 Microscopy and GFP fluorescence analysis ....................... 35
2.7 Constructions of the plasmids and strains ................................................................. 36
2.7.1 Construction of terminator-test vectors .............................. 36
2.7.2 Vectors and strains for the overexpression of α-amylase ... 37
2.8 Transposon mutagenesis and construction of a modified transposon ....................... 40
2.8.1 Detection of mutants able to increase secretion of α-amylase ........................... 41
2.8.2 Construction of a modified transposon .............................................................. 41

3 Results ............................................................................................... 44
3.1 The effect of the artificial bicistronic operon and the use of sinIR transcriptional
terminator as a 3‟-stabilizing element .................................................................................. 44
3.1.1 The GFP fluorescence analysis in the artificial bicistronic operon .................... 46
3.1.2 The BgaB activity analysis in the artificial bicistronic operon .......................... 47
3.2 The expression of α-amylase in B. subtilis by pKL01 ............................................... 50
3.2.1 Overexpression of B. subtilis secA does not improve secretion of α-amylase ... 53
3.2.2 The artificial secYEG operon increases the amount of secreted α-amylase in B.
subtilis .....................