High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization

The major platform for high level recombinant protein production is based on genetically modified microorganisms like Escherichia coli (E. coli) due to its short dividing time, ability to use inexpensive substrates and additionally, its genetics is comparatively simple, well characterized and can be manipulated easily. Here, we investigated the possibilities of finding the best media for high cell density fermentation, by analyzing different media samples, focusing on improving fermentation techniques and recombinant protein production. Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria–Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively). However, in fed-batch fermentation processes when using MMM after 25 h cultivation, it was possible to yield an optical density (OD600) of 139 corresponding to 172 g/L of wet biomass was produced in a 30 L TV Techfors-S Infors HT fermenter, with a computer controlled nutrient supply (glucose as a carbon source) delivery system, indicating nearly 1.5 times that obtained from TB. Upon purification, a total of 1.65 mg/g of protein per gram cell biomass was obtained and the purified AviPure showed affinity for immunoglobulin. High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition. It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.Fil: Kangwa, Martin. Jacobs University; AlemaniaFil: Yelemane, Vikas. Jacobs University; AlemaniaFil: Polat, Ayse Nur. Jacobs University; AlemaniaFil: Gorrepati, Kanaka Durga Devi. Jacobs University; AlemaniaFil: Grasselli, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Fernández Lahore, Marcelo. Jacobs University; Alemani

Design and production of peptide-based scaffolds for bioengineering applications

Protein- and peptide-based affinity reagents have demonstrated a great potential in different bioengineering fields, including the identification and capture of target molecules with applications in purification and sensing. This work focused on the study and production of cyclic β-hairpin peptides and Odorant-Binding Proteins (OBPs) as affinity reagents for application in bioseparation and biosensing, respectively. Two cyclic β-hairpin peptides (cyclic-M3 and cyclic-M9) were previously designed by docking, as potential affinity reagents for phosphorylated peptides. Here, cyclic-M3 and cyclic-M9, as well as a control peptide cyclic-M0 were chemically synthetized and characterized through Mass Spectrometry, analytical HPLC and Circular Dichroism. To evaluate the binding affinity of cyclic peptides towards several phosphorylated peptides, binding studies were performed in solution, by the MicroScale Thermophoresis technique. Cyclic-M3 and cyclic-M9 interact with a phosphorylated peptide GK14P with KA of 1.0 mM-1 and 1.34 mM-1, respectively. In addition, the cyclic peptides were selective for the phosphorylated moieties. Two rat OBPs (OBP2 and OBP3) were selected as experimental models for developing affinity reagents capable to detect specific volatile organic compounds (VOCs). Binding studies published until May 2018 reporting proteins selectivity and structural information were used to analyze structural characteristics involved in the natural binding of VOCs. Due to the lack in structural information for OBP2, homology modeling was employed to set a 3D structure. OBPs bind molecules with variable chemical and structural features mostly though hydrophobic interactions. However, the presence of determinant amino acid residues in the binding pockets increase the specificity of these proteins against VOCs. Both OBPs were successfully produced as soluble proteins using the E. coli expression system for further purification and biochemical characterization

Structure and Property of Polymers and Biopolymers from Molecular Dynamic Simulations

Natural and synthetic polymers and biopolymers have been studied for a variety of applications in food emulsion, biopharmaceutical purification, tissue engineering, and biosensor. The structure and property of polymers and biopolymers are critically important to determine their functions. Molecular dynamics (MD) simulations have a unique advantage to explore the structure and property of polymers and biopolymers from the molecular level. In the dissertation, MD simulations were conducted to study the mechanisms of various biological and chemical processes controlled by polymers and biopolymers based on real-world experimental results. Seven heptapeptides have been screened from a peptide library in our earlier study of the antibody purification. They have substantial binding affinities to the Fc fragment of IgG. In Chapter 2, the binding mechanisms between seven heptapeptides and the Fc fragment have been investigated by protein-ligand docking, free energy calculation and MD simulations. It is the first time that glycan residues are found to be the binding pocket for small ligands. The novel binding pocket is different from the CBS binding site for protein A and protein G. We also found out that, the results of free energy calculations are in good agreement with the ELISA experiments. The thermos-responsive polymer, PVCL (poly(N-vinylcaprolactam)) was grafted on the surface of a membrane as the responsive hydrophobic chromatography for the protein purification in our earlier study. In Chapter 3, significant efforts have been devoted to develop the force field parameters for PVCL. The coil-to-globule conformational transition of PVCL has been successfully observed in MD simulation for the first time. The water dynamics analysis provides significant insights into the interaction between PVCL and water molecules. The novel statistical analysis of VCL ring conformations and the distribution along backbone also elucidate the steric requirement in the coil-to-globule transition. In Chapter 4, MD simulations were conducted to investigate the biocompatibility, energetics and interaction mechanisms between the PVCL polymer chains and bovine serum albumin (BSA) in 1M NaCl and aqueous solutions. Water structures surrounding the polymer chains and BSA as well as their hydrogen bonding, electrostatic and van der Waals interactions were determined. Significant insights were obtained on the effects of polymer hydration state, polymer chain length as well as the presence of salt ions on the protein­ligand interactions. A novel polymeric solid acid catalyst consisting of two polymer chains grafted on a substrate for biomass hydrolysis was successfully synthesized. A poly (styrene sulfonic acid) (PSSA) polymer chain is immobilized on a substrate and used to catalyze biomass hydrolysis. A neighboring poly (vinyl imidazolium chloride) ionic liquid (PIL) polymer chain is grafted to help solubilize lignocellulosic biomass and enhance the catalytic activity. To elucidate mechanistically the catalytic actions and further optimize its performance, interactions among the PSSA, PIL, and cellulose chains were investigated using MD simulations in Chapter 5. Moreover, the free energies surfaces for the interactions between polymer chains and cellulose substrate were determined using combined MD and Metadynamics (MTD) simulations. The research clearly demonstrate that the solvent plays a critical role in the cellulose hydrolysis reaction catalyzed by novel enzyme mimic polymeric catalysts PSSA and PIL. It is found that PSSA chain is likely to form partially dehydrated interaction with cellulose in both aqueous and [EMIM]Cl solutions. PIL plays an important role to prevent the completely dehydrated interactions and facilitate partially dehydrated interaction between PSSA and cellulose chains