New Expression Method and Characterization of Recombinant Human Granulocyte Colony Stimulating Factor in a Stable Protein Formulation

Human recombinant granulocyte colony stimulating factor (rhG-CSF) is widely used in hematology and oncology for the treatment of neutropenia, for the restoration of neutrophil production after bone marrow transplantation, for myelodysplastic syndromes, and aplastic anemia. The E. coli expression system is commonly used for fast recombinant production of rhG-CSF at a large scale. We have applied a novel autoinduction method for the batch expression of rhG-CSF to study whether this new system would increase cell mass and target-protein yield compared to conventional E. coli cell culture and induction with isopropyl ?-D-thiogalactopyranoside (IPTG). We could demonstrate 3-fold higher culture densities and a 5-fold higher protein yield compared to IPTG induction without the need to monitor cell growth in a shortened 24 h expression procedure. rhG-CSF expressed in autoinduction media was successfully extracted from E. coli inclusion bodies and refolded by dialysis. After size exclusion chromatography (SEC) purification, rhG-CSF showed similar conformation, biological activity and aggregation profile compared to the commercially available biosimilar TEVAgrastim® (TEVA Pharma AG). Expression by autoinduction is suggested as a cost- and time-effective method for rhG-CSF production

Understanding tyrosine kinase domain plasticity through identification of protein residues involved in the control of the conformational transition

The aim of this thesis consisted in the identification of key amino acids residues governing the transition from active to inactive protein kinase domain conformation. We used the kinase domain of c-Src as a case study because of the wealth of structural information on this protein. We could demonstrate that residues governing the kinase domain dynamics of c-Src are located far from the ATP-binding site and form a network of hydrophobic interactions at the interface between the N-lobe and the C-lobe of the kinase domain. The identified amino acids and in particular Leu317 dictate the conformational plasticity of the kinase domain by influencing the rate of the DFG-flip. Furthermore, we could provide experimental evidence that the DFG-flip is the most probable molecular mechanism for the release of ADP at the end of the catalytic cycle and that both DFG-in and DFG-out conformations are present within the catalytic cycle of c-Src

The Different Flexibility of c-Src and c-Abl Kinases Regulates the Accessibility of a Druggable Inactive Conformation

c-Src and c-Abl are two closely related protein kinases that constitute important anticancer targets. Despite their high sequence identity, they show different sensitivities to the anticancer drug imatinib, which binds specifically to a particular inactive conformation in which the Asp of the conserved DFG motif points outward (DFG-out). We have analyzed the DFG conformational transition of the two kinases using massive molecular dynamics simulations, free energy calculations, and isothermal titration calorimetry. On the basis of the reconstruction of the free energy surfaces for the DFG-in to DFG-out conformational changes of c-Src and c-Abl, we propose that the different flexibility of the two kinases results in a different stability of the DFG-out conformation and might be the main determinant of imatinib selectivity

The Different Flexibility of c-Src and c-Abl Kinases Regulates the Accessibility of a Druggable Inactive Conformation

c-Src and c-Abl are two closely related protein kinases that constitute important anticancer targets. Despite their high sequence identity, they show different sensitivities to the anticancer drug imatinib, which binds specifically to a particular inactive conformation in which the Asp of the conserved DFG motif points outward (DFG-out). We have analyzed the DFG conformational transition of the two kinases using massive molecular dynamics simulations, free energy calculations, and isothermal titration calorimetry. On the basis of the reconstruction of the free energy surfaces for the DFG-in to DFG-out conformational changes of c-Src and c-Abl, we propose that the different flexibility of the two kinases results in a different stability of the DFG-out conformation and might be the main determinant of imatinib selectivity

The Different Flexibility of c-Src and c-Abl Kinases Regulates the Accessibility of a Druggable Inactive Conformation

c-Src and c-Abl are two closely related protein kinases that constitute important anticancer targets. Despite their high sequence identity, they show different sensitivities to the anticancer drug imatinib, which binds specifically to a particular inactive conformation in which the Asp of the conserved DFG motif points outward (DFG-out). We have analyzed the DFG conformational transition of the two kinases using massive molecular dynamics simulations, free energy calculations, and isothermal titration calorimetry. On the basis of the reconstruction of the free energy surfaces for the DFG-in to DFG-out conformational changes of c-Src and c-Abl, we propose that the different flexibility of the two kinases results in a different stability of the DFG-out conformation and might be the main determinant of imatinib selectivity