Functional monolithic platforms for antibody purification

Dissertação para obtenção do Grau de Doutor em Química SustentávelFundação para a Ciência e Tecnologia - contracts PEst-C/EQB/LA0006/2011, MIT-Pt/BS-CTRM/0051/2008, PTDC/EBB-BIO/102163/2008, PTDC/EBBBIO/ 098961/2008, PTDC/EBB-BIO/118317/2010 and doctoral grant SFRH/ BD/62475/2009, and Fundação Calouste Gulbenkia

An albumin-derived peptide scaffold capable of binding and catalysis

We have identified a 101-amino-acid polypeptide derived from the sequence surrounding the IIA binding site of human albumin. The polypeptide contains residues that make contact with ligands as warfarin in the parent protein, and eight cysteine residues to form disulfide bridges, which stabilize the polypeptide structure. Seventy-four amino acids are located in six [alpha]-helical regions, with the remaining amino acids forming six connecting coil/loop regions. Codon usage optimization was used to express a GST fusion protein in E. coli in yields as high as 4 mg/l. This fusion protein retains its structural integrity and aldolase activity, the ability to direct the stereochemical outcome of a diketone reduction, and its binding capacity to warfarin and efavirenz. Notably, this newly cloned polypeptide represents a valuable starting point for the construction of libraries of binders and catalysts with improved proficiency

Inspirations of biomimetic affinity ligands: a review

Affinity chromatography is a well-known method dependent on molecular recognition and is used to purify biomolecules by mimicking the specific interactions between the biomolecules and their substrates. Enzyme substrates, cofactors, antigens, and inhibitors are generally utilized as bioligands in affinity chromatography. However, their cost, instability, and leakage problems are the main drawbacks of these bioligands. Biomimetic affinity ligands can recognize their target molecules with high selectivity. Their cost-effectiveness and chemical and biological stabilities make these antibody analogs favorable candidates for affinity chromatography applications. Biomimetics applies to nature and aims to develop nanodevices, processes, and nanomaterials. Today, biomimetics provides a design approach to the biomimetic affinity ligands with the aid of computational methods, rational design, and other approaches to meet the requirements of the bioligands and improve the downstream process. This review highlighted the recent trends in designing biomimetic affinity ligands and summarized their binding interactions with the target molecules with computational approaches

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

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

Trends in the design and use of elastin-like recombinamers as biomaterials

Producción CientíficaElastin-like recombinamers (ELRs), which derive from one of the repetitive domains found in natural elastin, have been intensively studied in the last few years from several points of view. In this mini review, we discuss all the recent works related to the investigation of ELRs, starting with those that define these polypeptides as model intrinsically disordered proteins or regions (IDPs or IDRs) and its relevance for some biomedical applications. Furthermore, we summarize the current knowledge on the development of drug, vaccine and gene delivery systems based on ELRs, while also emphasizing the use of ELR-based hydrogels in tissue engineering and regenerative medicine (TERM). Finally, we show different studies that explore applications in other fields, and several examples that describe biomaterial blends in which ELRs have a key role. This review aims to give an overview of the recent advances regarding ELRs and to encourage further investigation of their properties and applications.Comisión Europea (project NMP-2014-646075)Ministerio de Economía, Industria y Competitividad (projects PCIN-2015-010 / MAT2016-78903-R / BES-2014-069763)Junta de Castilla y León (project VA317P18

Magnetic nanoparticles as a versatile solid-support for fusion protein purification and antimicrobial assays

Magnetic nano-and microparticles are unique platforms for the development of bioseparation and antimicrobial devices. This work explored the application of magnetic particles for the purification of fusion proteins through the use of magnetic adsorbents coupled to novel affinity ligands towards peptidic and proteic tags. Furthermore, and in view of the novelty of these ligands, molecular modeling and simulation techniques were employed to explain the key structuralfeatures involved inthe binding of two affinity pairs: GFP/LA-A4C7 and RK-GFP/LR-A7C1.The results showed that the interaction between GFP and LA-A4C7 is mainly hydrophobicwhile the interaction between RK-GFP and LR-A7C1 is mostly driven byhydrogen bonds. Moreover, the same modeling techniques have been used to idealize a theoretical second generation library with view of maximizing the estimated free energy of binding and the correspondent affinity constant. When immobilizing the biomimetic ligands LA-A4C7 and LR-A7C1 onto magnetic nanoparticles, it was possibleto bind the protein of interest and recover pure elution fractions. The best elution condition for GFP elution was 0.1mM glycine-NaOH pH9 50% (v/v) ethylene glycoland the best elution condition for RK-GFP elution was PBS pH 7.4, 500mM arginine, which are in accordance with the theoretical results described previously. Final binding constants for the studied systems (Ka=0.83×105M-1and Qmax=4mg/g for GFP/LA-A4C7, Ka=3.21×105M-1and Qmax=2mg/g for RK-GFP/LR-A7C1) show promising results for an affinity-based protein purification system.Magnetic particleswere also functionalized with (RW)3, an peptidewith antimicrobial properties, by different routes. We were able to develop a novel antimicrobial nanodevice based on the EDC-coupling of (RW)3that has shown antimicrobial activity against Escherichiacoliand Bacillussubtilis

Novel affinity pairs "tag-receptor" for the purification of fusion proteins

Fundação para a Ciência e Tecnologia - SFRH/BD/48804/2008 and the project PTDC/BI/65383/2006 assigned to Prof. Cecíla Roque and also to Associate Laboratory REQUIMTE (Pest-C/EQB/LA0006/2011