Müller, Jakob: Development of methods for the production and purification of streptavidin from Streptomyces avidinii and heterologous hosts. 2016
Inhalt
- List of Figures
- List of Tables
- Abstract
- Introduction
- Binding of biotin to streptavidin (SAV)
- Properties and applications of SAV
- Other biotin-binding proteins
- Homologous and heterologous production of SAV
- Homologous production of SAV
- Heterologous production of SAV
- Product toxicity for heterologous expression of the SAV gene
- Purification of SAV
- Focus of this dissertation
- Software
- Abbreviations
- Structural remarks
- Development of an assay for the detection of biotin-blocked binding sites of SAV
- Motivation
- Concept of the assay
- Manuscript
- Publication: Model-based development of an assay for the rapid detection of biotin-blocked binding sites of SAV
- Supplementary material to the publication
- Potential applications for the assay
- Recovery of biotin-blocked binding sites of SAV
- Production of SAV by Streptomyces avidinii
- Motivation
- Manuscript
- Publication: Development of fed-batch-strategies for the production of SAV by S. avidinii
- Corrigendum to 'Development of fed-batch-strategies for the production of SAV by S. avidinii'
- Theoretical analysis of the accuracy of the power input measurements
- Dimensions of the MBR bioreactor.
- Reynolds number and power equation
- Performance characteristics of different stirrers
- System-based modifications
- Calculation of flow regime and power input
- Estimation of aerated power input
- Comparison of experimental and theoretical values
- Load-dependent efficiency of electric motors - an error analysis
- Correlation of kLa and Lspec
- Power input and streptomycetes in literature
- Summary of additional experiments applying S. avidinii
- Heterologous expression of the SAV gene
- Motivation
- Production of SAV by Escherichia coli
- Goals of this study
- Manuscript
- Publication: Constitutive production and efficient secretion of soluble full-length SAV by an E. coli 'leaky mutant'
- Supplementary material to the publication
- Additional information: coexpression of inhibitors
- Production of SAV by Pichia pastoris
- Goals of this study
- Manuscript
- Publication: GAP promoter-based fed-batch production of highly bioactive core SAV by P. pastoris
- Supplementary material to the publication
- Summary of additional experiments applying P. pastoris
- Production of SAV by Hansenula polymorpha
- Evaluation of alternative methods for the purification of SAV
- Motivation
- Host-dependent product purity
- Summary of the results
- Aqueous two phase extraction
- Filtration
- Ammonium sulfate precipitation
- Thermal inactivation of contaminating proteins
- Suitability of different methods of purification
- Summary, concluding remarks and outlook
- Comparative analysis of SAV production by four different hosts
- Comparison of the overall performance
- Discussion of the specific productivity
- Analysis of process robustness
- Genetic stability of the SAV expression cassette
- Effect of the cultivation temperature
- Effect of the secretory potential of the host
- Suitability of yeast-based expression systems for the production of SAV
- Analysis of product purity
- Ecological assessment
- Summary of the qualitative host-specific properties
- Comparison of the results to literature
- Conclusions and outlook
- Supplementary material
- Additional methods
- Recovery of biotin-blocked binding sites
- Homologous production of SAV by S. avidinii
- Purification of SAV
- Supporting results
- In silico-analysis of the native signal peptide of SAV from S. avidinii
- Recovery of biotin-blocked binding sites of SAV
- Course of the dynamic viscosity throughout a cultivation of S. avidinii
- Power input for the studies of S. avidinii
- Additional figures
- Bibliography