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

Bioinspired and Sustainable Chitosan Based Monoliths for Antibody Capture and Release

The authors would like to thank the financial support from Fundacao para a Ciencia e Tecnologia, Portugal, through contracts MIT-Pt/BS-CTRM/0051/2008, PTDC/EBB-BIO/102163/2008, PTDC/EBB-BIO/098961/2008 and PTDC/EBB-BIO/118317/2010 and doctoral grant SFRH/BD/62475/2009 (T. B.), Bioengineering Systems Focus Area, Fundacao Calouste Gulbenkian, FEDER and FSE. We wish to thank the Analytical Services Laboratory of REQUIMTE for the characterization of materials. Authors are also thankful to Lonza Biologics, UK (Dr Richard Alldread) and the Animal Cell Technology Unit of ITQB-UNL/IBET (Dr Paula M Alves and Dr Ana Teixeira) for providing the cell culture bulks of antibodies.Chitosan-based monoliths activated by plasma technology induced the coupling of a robust biomimetic ligand, previously reported as an artificial Protein A, with high yields while minimizing the environmental impact of the procedure. Due to the high porosity, good mechanical and tunable physicochemical properties of the affinity chitosan-based monoliths, it is possible to achieve high binding capacities (150 ± 10 mg antibody per gram support), and to recover 90 ± 5% of the bound protein with 98% purity directly from cell-culture extracts. Therefore, the chitosan-based monoliths prepared by clean processes exhibit a remarkable performance for the one-step capture and recovery of pure antibodies or other biological molecules with biopharmaceutical relevance.authorsversionpublishe

Seeing the Unseen: The Role of Liquid Crystals in Gas-Sensing Technologies

SCENT-ERC-2014-STG-639123 (2015-2020) UIDB/04378/2020 SFRH/BD/113112/2015Fast, real-time detection of gases and volatile organic compounds (VOCs) is an emerging research field relevant to most aspects of modern society, from households to health facilities, industrial units, and military environments. Sensor features such as high sensitivity, selectivity, fast response, and low energy consumption are essential. Liquid crystal (LC)-based sensors fulfill these requirements due to their chemical diversity, inherent self-assembly potential, and reversible molecular order, resulting in tunable stimuli-responsive soft materials. Sensing platforms utilizing thermotropic uniaxial systems—nematic and smectic—that exploit not only interfacial phenomena, but also changes in the LC bulk, are demonstrated. Special focus is given to the different interaction mechanisms and tuned selectivity toward gas and VOC analytes. Furthermore, the different experimental methods used to transduce the presence of chemical analytes into macroscopic signals are discussed and detailed examples are provided. Future perspectives and trends in the field, in particular the opportunities for LC-based advanced materials in artificial olfaction, are also discussed.publishersversionpublishe

Antibody-Conjugated Nanoparticles for Therapeutic Applications

Ines Peca is grateful for financial support from Fundacao para a Ciencia e a Tecnologia through contract SFRH/BD/48773/2008. This work has been supported by Fundacao para a Ciencia e a Tecnologia through grant no. PEst-C/EQB/LA0006/2011.A great challenge to clinical development is the delivery of chemotherapeutic agents, known to cause severe toxic effects, directly to diseased sites which increase the therapeutic index whilst minimizing off-target side effects. Antibody-conjugated nanoparticles offer great opportunities to overcome these limitations in therapeutics. They combine the advantages given by the nanoparticles with the ability to bind to their target with high affinity and improve cell penetration given by the antibodies. This specialized vehicle, that can encapsulate several chemotherapeutic agents, can be engineered to possess the desirable properties, allowing overcoming the successive physiological conditions and to cross biological barriers and reach a specific tissue or cell. Moreover, antibody-conjugated nanoparticles have shown the ability to be internalized through receptor-mediated endocytosis and accumulate in cells without being recognized by the P-glycoprotein, one of the main mediators of multi-drug resistance, resulting in an increase in the intracellular concentration of drugs. Also, progress in antibody engineering has allowed the manipulation of the basic antibody structure for raising and tailoring specificity and functionality. This review explores recent developments on active drug targeting by nanoparticles functionalized with monoclonal antibodies (polymeric micelles, liposomes and polymeric nanoparticles) and summarizes the opportunities of these targeting strategies in the therapy of serious diseases (cancer, inflammatory diseases, infectious diseases, and thrombosis).authorsversionpublishe

Impact of the Cationic Moiety of Ionic Liquids on Chemoselective Artificial Olfaction

Funding Information: This research was funded by the European Research Council (ERC) under the EU Horizon 2020 research and innovation program [grant reference SCENT-ERC-2014-STG-639123, 2015–2022, and Grant Agreement No. 101069405─ENSURE─ERC-2022-POC1] and by national funds from FCT-Fundação para a Ciência e a Tecnologia, I.P., for projects PTDC/BII-BIO/28878/2017, PTDC/CTM-CTM/3389/2021, UIDP/04378/2020, and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences-UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy-i4HB. The authors thank FCT/MCTES for the PhD grant SFRH/BD/128687/2017. Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.Ionogels and derived materials are assemblies of polymers and ionic liquids characterized by high stability and ionic conductivity, making them interesting choices as gas sensors. In this work, we assessed the effect of the ionic liquid moiety to generate ionogels and hybrid gels as electrical and optical gas sensors. Six ionic liquids consisting of a constant anion (chloride) and distinct cationic head groups were used to generate ionogels and hybrid gels and further tested as gas sensors in customized electronic nose devices. In general, ionogel-based sensors yielded higher classification accuracies of standard volatile organic compounds when compared to hybrid material-based sensors. In addition, the high chemical diversity of ionic liquids is further translated to a high functional diversity in analyte molecular recognition and sensing.publishersversionpublishe

Sustainable plant polyesters as substrates for optical gas sensors

UIDB/04378/2020 PTDC/BII-BIO/28878/2017 PTDC/SAU-SER/30388/2017 SFRH-BD-110467-2015The fast and non-invasive detection of odors and volatile organic compounds (VOCs) by gas sensors and electronic noses is a growing field of interest, mostly due to a large scope of potential applications. Additional drivers for the expansion of the field include the development of alternative and sustainable sensing materials. The discovery that isolated cross-linked polymeric structures of suberin spontaneously self-assemble as a film inspired us to develop new sensing composite materials consisting of suberin and a liquid crystal (LC). Due to their stimuli-responsive and optically active nature, liquid crystals are interesting probes in gas sensing. Herein, we report the isolation and the chemical characterization of two suberin types (from cork and from potato peels) resorting to analyses of gas chromatography–mass spectrometry (GC-MS), solution nuclear magnetic resonance (NMR), and X-ray photoelectron spectroscopy (XPS). The collected data highlighted their compositional and structural differences. Cork suberin showed a higher proportion of longer aliphatic constituents and is more esterified than potato suberin. Accordingly, when casted it formed films with larger surface irregularities and a higher C/O ratio. When either type of suberin was combined with the liquid crystal 5CB, the ensuing hybrid materials showed distinctive morphological and sensing properties towards a set of 12 VOCs (comprising heptane, hexane, chloroform, toluene, dichlormethane, diethylether, ethyl acetate, acetonitrile, acetone, ethanol, methanol, and acetic acid). The optical responses generated by the materials are reversible and reproducible, showing stability for 3 weeks. The individual VOC-sensing responses of the two hybrid materials are discussed taking as basis the chemistry of each suberin type. A support vector machines (SVM) algorithm based on the features of the optical responses was implemented to assess the VOC identification ability of the materials, revealing that the two distinct suberin-based sensors complement each other, since they selectively identify distinct VOCs or VOC groups. It is expected that such new environmentally-friendly gas sensing materials derived from natural diversity can be combined in arrays to enlarge selectivity and sensing capacity.publishersversionpublishe

Solvent modulation in peptide sub-microfibers obtained by solution blow spinning

Peptides possess high chemical diversity at the amino acid sequence level, which further translates into versatile functions. Peptides with self-assembling properties can be processed into diverse formats giving rise to bio-based materials. Peptide-based spun fibers are an interesting format due to high surface-area and versatility, though the field is still in its infancy due to the challenges in applying the synthetic polymer spinning processes to protein fibers to peptides. In this work we show the use of solution blow-spinning to produce peptide fibers. Peptide fiber formation was assisted by the polymer poly (vinyl pyrrolidone) (PVP) in two solvent conditions. Peptide miscibility and further self-assembling propensity in the solvents played a major role in fiber formation. When employing acetic acid as solvent, peptide fibers (0.5 μm) are formed around PVP fibers (0.75 μm), whereas in isopropanol only one type of fibers are formed, consisting of mixed peptide and PVP (1 μm). This report highlights solvent modulation as a mean to obtain different peptide sub-microfibers via a single injection nozzle in solution blow spinning. We anticipate this strategy to be applied to other small peptides with self-assembly propensity to obtain multi-functional proteinaceous fibers.ISSN:2296-264

A purification platform for antibodies and derived fragments using a de novo designed affinity adsorbent

Fundação para a Ciência e Tecnologia (FCT) -PTDC/BII-BIO/28878/2017; co-financed by ERDF under the PT2020 Partnership Agreement LISBOA-01-0145-FEDER-028878; PD/BD/128251/2016 from MIT-PT PhD Bioengineering Systems to MJBM; SFRH/BPD/112543/2015 to AJMB; Applied Molecular Biosciences Unit – UCIBIO (UIDP/04378/2020 ; UIDB/04378/2020); CICECO-Aveiro Institute of Materials (UIDB/50011/2020; UIDP/50011/2020), financed by national funds from FCT.Antibody technologies are the most representative success-case in the biopharmaceutical industry. Widely available purification technologies fail in providing a dedicated universal purification platform that can accommodate antibodies structural diversity, namely antibodies from non-human sources, as chicken IgY, and antigen-binding fragments. In this work, we took inspiration from natural and engineered antibody-binding ligands, to rationally design affinity adsorbents able to capture full-length antibodies and fragments. The one-pot Petasis and Ugi combinatorial reactions were sequentially employed to rapidly generate a library of putative solid-phase adsorbents. The best performing adsorbent yielded a single-step recovery, under mild conditions, of human and chicken whole antibodies, antigen-binding fragments and engineered single-domain antibodies from different complex feedstocks. Due to its simple preparation, the lead antibody adsorbent finds broad applicability as a universal purification platform to increase the availability of antibody technologies in research and development.authorsversionpublishe

DataSheet1_Solvent modulation in peptide sub-microfibers obtained by solution blow spinning.docx

Peptides possess high chemical diversity at the amino acid sequence level, which further translates into versatile functions. Peptides with self-assembling properties can be processed into diverse formats giving rise to bio-based materials. Peptide-based spun fibers are an interesting format due to high surface-area and versatility, though the field is still in its infancy due to the challenges in applying the synthetic polymer spinning processes to protein fibers to peptides. In this work we show the use of solution blow-spinning to produce peptide fibers. Peptide fiber formation was assisted by the polymer poly (vinyl pyrrolidone) (PVP) in two solvent conditions. Peptide miscibility and further self-assembling propensity in the solvents played a major role in fiber formation. When employing acetic acid as solvent, peptide fibers (0.5 μm) are formed around PVP fibers (0.75 μm), whereas in isopropanol only one type of fibers are formed, consisting of mixed peptide and PVP (1 μm). This report highlights solvent modulation as a mean to obtain different peptide sub-microfibers via a single injection nozzle in solution blow spinning. We anticipate this strategy to be applied to other small peptides with self-assembly propensity to obtain multi-functional proteinaceous fibers.</p

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