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

A scalable method to purify reflectins from inclusion bodies

Associate Laboratory Institute for Health and Bioeconomy – i4HB (LA/P/0140/2020). 2022.11305.BD for C.S. Publisher Copyright: © 2023 The Author(s)Structural proteins are an attractive inspiration for functional biobased materials. In nature, cephalopods skin colour modulation is related to the dynamic self-assembly of a family of structural proteins known as reflectins. To fully reach their potential as engineered bio-based materials, reflectins need to be produced by biotechnological means. One of the challenges is associated with establishing and optimizing reflectin purification processes to achieve the highest yield and productivity. Here, we studied purification strategies for two reflectin sequences from different organisms which were recombinantly expressed in a bacterial host at laboratory scale. Reflectins purification was then assessed by two chromatographic and one non-chromatographic methods. Methods were compared considering final purity and yield, productivity, cost and sustainability. The non-chromatographic method based on inclusion bodies washing presented the most promising results (protein purity > 90% and purification yields up to 88%). Our results contribute to define bioprocessing strategies to address the vision of biodegradable and sustainable protein-based materials.publishersversionpublishe

Identification and Antibiotic-Susceptibility Profiling of Infectious Bacterial Agents: A Review of Current and Future Trends

This work was supported by the European Research Council through the grant reference SCENT-ERC-2014-STG-639123 (2015-2020), and by the Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO, which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728).Antimicrobial resistance is one of the most worrying threats to humankind with extremely high healthcare costs associated. The current technologies used in clinical microbiology to identify the bacterial agent and profile antimicrobial susceptibility are time-consuming and frequently expensive. As a result, physicians prescribe empirical antimicrobial therapies. This scenario is often the cause of therapeutic failures, causing higher mortality rates and healthcare costs, as well as the emergence and spread of antibiotic resistant bacteria. As such, new technologies for rapid identification of the pathogen and antimicrobial susceptibility testing are needed. This review summarizes the current technologies, and the promising emerging and future alternatives for the identification and profiling of antimicrobial resistance bacterial agents, which are expected to revolutionize the field of clinical diagnostics.publishersversionpublishe

Hierarchical self-assembly of a reflectin-derived peptide

Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin’s basic unit—the protopeptide sequence YMDMSGYQ—as a means to understand reflectin’s assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.ISSN:2296-264

DataSheet1_Hierarchical self-assembly of a reflectin-derived peptide.docx

Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin’s basic unit—the protopeptide sequence YMDMSGYQ—as a means to understand reflectin’s assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.</p