Entwicklung einer neuen funktionellen Proteintechnologie in E. coli

Abstract

The aim of this Ph.D thesis was the establishment of a novel Functional Protein Technology (FunProTec). It is based upon the combined application of a pro- or eukaryotic expression and secretion system, that allows a quick and easy high yield synthesis of native protein solutions or protein arrays consisting of functionally active proteins, and a compartment system, which preserves the respective proteins from the producing cells and separates them from background proteins. FunProTec is an invention of ALTANA Pharma and was patented with the international publication number WO 02/50260 A2, international publication date 27.06.2002.In this study, FunProTec was established by utilization of a modified version of the E. coli alpha-hemolysin secretion system. The hemolysin secretion apparatus consists of the proteins HlyB, HlyD and TolC and can be utilized for the secretion of heterologous proteins by generating gene fusions between heterologous genes and the C-terminal signal sequence HlyAs of hemolysin (Gentschev et al., 1994).In this thesis, a system for synthesis and secretion of functional active proteins was validated by generating different E. coli strains suitable for the secretion of heterologous proteins by the hemolysin secretion system. The genes encoding the transport proteins HlyB and HlyD and the genes encoding hemolysin (HlyA) and its activator protein (HlyC) were cloned either onto the same (case A) or onto two different plasmids (case B) in these E. coli strains, while the tolC gene was located on the bacterial chromosome. These E. coli strains were compared in regard to their hemolysin secretion efficiencies.It was demonstrated, that in both cases (A and B) a DNA sequence located upstream of the hly genes is essential for the hemolytic activity of the respective E. coli strain and can not be displaced by a heterologous promoter. This sequence is also located upstream of the wild-type hemolysin gene cluster in the chromosome of uropathogenic E. coli. It contains the hemolysin promoter and a JUMPstart activator sequence (Hobbs and Reeves, 1994).It was also demonstrated in this study that a defined stoichiometry between the transcripts of the genes encoding hemolysin (hlyA) and the transcripts encoding the transport channel hlyB and hlyD is responsible for efficient secretion of hemolysin and is regulated by the above mentioned activator sequence. This indicates that the hemolysin operon is regulated in a complex manner and that every genetic manipulation may influence the transcription of the hly genes and possibly the secretion efficiency of the respective E. coli strain.In the above described E. coli strains A and B, the secretion efficacies of alkaline phosphatase by E. coli K-12 strains, the E. coli outer membrane protein A (OmpA), the human phosphodiesterase 1B1 and the human cytokine receptor ligand Ccl-21 were examined. None of these proteins was secreted. The secretion of heterologous proteins by the hemolysin secretion system has been described, however, with the exception of protein toxins or very small proteins or peptides, only two full-length proteins were secreted. It remains to be investigated that the loss of protein folding may facilitate the secretion. It has to be further investigated whether the application of chaperons that change the protein folding will improve protein secretion.The present study suggest, that the hemolysin secretion system is not a universal secretion system, but it might be suitable for secretion of small, unfolded proteins as shown by the secretion of protein subunits and antibody chains (Tzatschel et al., 1996, Fernandez et al., 2000).Moreover, the compartment systems were validated in this thesis by showing that the hemolysin protein was separated from the secreting E. coli strains by suitable filter systems without loosing its activity.In conclusion, FunProTec may be used for different pro- or eukaryotic secretion systems. Its applicability depends on the properties of the protein to be secreted.Thus, in combination with the corresponding compartment systems, FunProTec might be a suitable technology for production of recombinant proteins.