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Dias, AMGC, dos Santos R, Iranzo O, Roque ACA.  2016.  Affinity adsorbents for proline-rich peptide sequences: a new role for WW domains. RSC Adv.. 6:68979-68988.: The Royal Society of Chemistry AbstractWebsite

The WW domain derived from human Yes-associated protein (hYAP65_WW) recognizes proline-rich peptides. The structural and chemical robustness of WW domains makes them appealing candidates to target and capture these peptides in affinity purification processes. In this work{,} the chemical synthesis of the hYAP65_WW domain containing a terminal cysteine for oriented coupling onto the chromatographic matrix was successfully achieved by a fragment solution condensation reaction and by incorporation of pseudoproline dipeptide units. Both strategies yielded a hYAP65_WW protein with the characteristic WW domain folding. The purified hYAP65_WW domain was immobilized in a chromatographic matrix and tested for binding to a proline-rich peptide. The adsorbent bound 92 ng of peptide per mg of support and the elution was particularly efficient when employing a low pH or an increase in salt concentration. This work sets the ground for the application of WW domains as affinity reagents towards the capture and elution of peptides and proteins rich in proline sequences.

Pina, AS, Batalha IL, Dias AMGC, Roque ACA.  2021.  Affinity tags in protein purification and peptide enrichment: An overview, in Protein Downstream Processing: Design, Development, and Application of High and Low-Resolution Methods. Methods in Molecular Biology. :107-132.: Springer-Humana Press
Fernandes, C, Pina AS, Barbosa AJM, Padrão I, Duarte F, Andreia C, Teixeira S, Alves V, Gomes P, Fernandes TG, Dias AMGC, Roque ACA.  2019.  Affinity‐triggered assemblies based on a designed peptide‐peptide affinity pair. Biotechnology Journal. -(-):-. AbstractWebsite

Affinity‐triggered assemblies rely on affinity interactions as the driving force to assemble physically‐crosslinked networks. WW domains are small hydrophobic proteins binding to proline‐rich peptides that are typically produced in the insoluble form. Previous works attempted the biological production of the full WW domain in tandem to generate multivalent components for affinity‐triggered hydrogels. In this work, an alternative approach was followed by engineering a 13‐mer minimal version of the WW domain that retains the ability to bind to target proline‐rich peptides. Both ligand and target peptides were produced chemically and conjugated to multivalent polyethylene glycol, yielding two components. Upon mixing, they together form soft biocompatible affinity‐triggered assemblies, stable in stem cell culture media, and displaying mechanical properties in the same order of magnitude as for those hydrogels formed with the full WW protein in tandem.

Dias, AMGC, Hussain A, Marcos AS, Roque ACA.  2011.  A biotechnological perspective on the application of iron oxide magnetic colloids modified with polysaccharides. Biotechnology Advances. 29:142–155., Number 1 AbstractWebsite

Iron oxide magnetic nanoparticles {(MNPs)} alone are suitable for a broad spectrum of applications, but the low stability and heterogeneous size distribution in aqueous medium represent major setbacks. These setbacks can however be reduced or diminished through the coating of {MNPs} with various polymers, especially biopolymers such as polysaccharides. Polysaccharides are biocompatible, non-toxic and renewable; in addition, they possess chemical groups that permit further functionalization of the {MNPs.} Multifunctional entities can be created through decoration with specific molecules e.g. proteins, peptides, drugs, antibodies, biomimetic ligands, transfection agents, cells, and other ligands. This development opens a whole range of applications for iron oxide nanoparticles. In this review the properties of magnetic structures composed of {MNPs} and several polysaccharides {(Agarose}, Alginate, Carrageenan, Chitosan, Dextran, Heparin, Gum Arabic, Pullulan and Starch) will be discussed, in view of their recent and future biomedical and biotechnological applications.

Dhadge, VL, Hussain A, Azevedo AM, Aires-Barros MR, Roque ACA.  2014.  Boronic acid-modified magnetic materials for antibody purification. J. R. Soc. Interface. 11(91):20130875. AbstractWebsite

Aminophenyl boronic acids can form reversible covalent ester interactions with cis-diol-containing molecules, serving as a selective tool for binding glycoproteins as antibody molecules that possess oligosaccharides in both the Fv and Fc regions. In this study, amino phenyl boronic acid (APBA) magnetic particles (MPs) were applied for the magnetic separation of antibody molecules. Iron oxide MPs were firstly coated with dextran to avoid non-specific binding and then with 3-glycidyloxypropyl trimethoxysilane to allow further covalent coupling of APBA (APBA_MP). When contacted with pure protein solutions of human IgG (hIgG) and bovine serum albumin (BSA), APBA_MP bound 170 ± 10 mg hIgG g−1 MP and eluted 160 ± 5 mg hIgG g−1 MP, while binding only 15 ± 5 mg BSA g−1 MP. The affinity constant for the interaction between hIgG and APBA_MP was estimated as 4.9 × 105 M−1 (Ka) with a theoretical maximum capacity of 492 mg hIgG adsorbed g−1 MP (Qmax), whereas control particles bound a negligible amount of hIgG and presented an estimated theoretical maximum capacity of 3.1 mg hIgG adsorbed g−1 MP (Qmax). APBA_MPs were also tested for antibody purification directly from CHO cell supernatants. The particles were able to bind 98% of IgG loaded and to recover 95% of pure IgG (purity greater than 98%) at extremely mild conditions.

Santana, SDF, Dhadge VL, Roque ACA.  2012.  Dextran-Coated Magnetic Supports Modified with a Biomimetic Ligand for IgG Purification. ACS Applied Materials and Interfaces. 4(11):5907–5914. AbstractWebsite

extran-coated iron oxide magnetic particles modified with ligand 22/8, a protein A mimetic ligand, were prepared and assessed for IgG purification. Dextran was chosen as the agent to modify the surface of magnetic particles by presenting a negligible level of nonspecific adsorption. For the functionalization of the particles with the affinity ligand toward antibodies, three methods have been explored. The optimum coupling method yielded a theoretical maximum capacity for human IgG calculated as 568 ± 33 mg/g and a binding affinity constant of 7.7 × 104 M–1. Regeneration, recycle and reuse of particles was also highly successful for five cycles with minor loss of capacity. Moreover, this support presented specificity and effectiveness for IgG adsorption and elution at pH 11 directly from crude extracts with a final purity of 95% in the eluted fraction.

Palma, SICJ, Esteves C, Pádua AC, Alves CM, Santos GMC, Costa HMA, Dionisio M, Gamboa H, Gruber J, Roque ACA.  2019.  Enhanced gas sensing with soft functional materials, May 2019. ISOEN 2019 - 18th International Symposium on Olfaction and Electronic Nose, Proceedings. , Fukuoka, Japan: Institute of Electrical and Electronics Engineers Inc. AbstractPDF

The materials described in this work result from the selfassembly of liquid crystals and ionic liquids into droplets,
stabilized within a biopolymeric matrix. These systems are
extremely versatile gels, in terms of composition, and offer
potential for fine tuning of both structure and function, as
each individual component can be varied. Here, the
characterization and application of these gels as sensing thin
films in gas sensor devices is presented. The unique
supramolecular structure of the gels is explored for molecular
recognition of volatile organic compounds (VOCs) by
employing gels with distinct formulations to yield
combinatorial optical and electrical responses used in the
distinction and identification of VOCs.

Dhadge, VL, Morgado PI, Freitas F, Reis MA, Azevedo AM, Aires-Barros R, Roque ACA.  2014.  An extracellular polymer at the interface of magnetic bioseparations. Journal of the Royal Society Interface. 11(100):20140743. AbstractWebsite

FucoPol, a fucose-containing extracellular polysaccharide (EPS) produced by bacterium Enterobacter A47 using glycerol as the carbon source, was employed as a coating material for magnetic particles (MPs), which were subsequently functionalized with an artificial ligand for the capture of antibodies. The performance of the modified MPs (MP–EPS-22/8) for antibody purification was investigated using direct magnetic separation alone or combined with an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and dextran. In direct magnetic capturing, and using pure protein solutions of human immunoglobulin G (hIgG) and bovine serum albumin (BSA), MP–EPS-22/8 bound 120 mg hIgG g−1 MPs, whereas with BSA only 10 ± 2 mg BSA g−1 MPs was achieved. The hybrid process combining both the ATPS and magnetic capturing leads to a good performance for partitioning of hIgG in the desired phase as well as recovery by the magnetic separator. The MPs were able to bind 145 mg of hIgG g−1 of particles which is quite high when compared with direct magnetic separation. The theoretical maximum capacity was calculated to be 410 ± 15 mg hIgG adsorbed g−1 MPs with a binding affinity constant of 4.3 × 104 M−1. In multiple extraction steps, the MPs bound 92% of loaded hIgG with a final purity level of 98.5%. The MPs could easily be regenerated, recycled and re-used for five cycles with only minor loss of capacity. FucoPol coating allowed both electrostatic and hydrophobic interactions with the antibody contributing to enhance the specificity for the targeted products.

Dias, AMGC, Roque ACA.  2017.  The future of protein scaffolds as affinity reagents for purification. Biotechnology and Bioengineering. 114:481–491., Number 3 Abstract

Affinity purification is one of the most powerful separation techniques extensively employed both at laboratory and production scales. While antibodies still represent the gold standard affinity reagents, others derived from non-immunoglobulin scaffolds emerged as interesting alternatives in particular for affinity purification. The lower costs of production, fast ligand development and high robustness are appealing advantages of non-immunoglobulin scaffolds. These have successfully been used in the affinity purification of relevant targets as antibodies, human serum albumin, transferrin and other biomarkers, as reviewed in this work. Furthermore, a critical assessment on the strengths, weaknesses, opportunities and threats related with the implementation of non-immunoglobulin scaffolds as ligands in affinity purification are discussed. This article is protected by copyright. All rights reserved.

Dias, AMGC, Iranzo O, Roque ACA.  2015.  An in silico and chemical approach towards small protein production and application in phosphoproteomics. RSC Adv.. 5:19743-19751.: The Royal Society of Chemistry AbstractWebsite

The human Pin1 WW domain (hPin1_WW) is a 38 residue protein which specifically recognizes ligands rich in proline and phosphorylated in Ser and Thr residues. This work presents a protocol for the improved chemical synthesis and modification of this protein through automated microwave assisted synthesis combined with the incorporation of pseudoproline units in the protein sequence. After purification{,} the protein was characterized by Mass Spectrometry and Circular Dichroism spectroscopy with results comparable to the same WW domain chemically synthesized by other strategies or biologically expressed. The protein was further immobilized on a matrix and tested for the selective binding and mild elution of phosphorylated sequences at Ser{,} Thr and Tyr residues. These results suggest that hPin1_WW is a useful protein scaffold for the purification of phosphorylated species in pTyr and pSer{,} which can be easily produced and modified by chemical methods.

Bicho, A, Roque ACA, Cardoso AS, Domingos P, Batalha ÍL.  2010.  In vitro studies with mammalian cell lines and gum arabic‐coated magnetic nanoparticles. Journal of Molecular Recognition. 23:536–542., Number 6 AbstractWebsite

Iron oxide magnetic nanoparticles {(MNPs)} were synthesized by the chemical co-precipitation method and coated with gum arabic {(GA)} by physical adsorption and covalent attachment. Cultures of mammalian cell lines {(HEK293}, {CHO} and {TE671)} were grown in the presence of uncoated and {GA-coated} {MNPs.} Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for {GA} coating of {MNPs} endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the {GA} was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures. Copyright © 2010 John Wiley & Sons, Ltd.

Dhadge, VL, Rosa S, Azevedo A, Aires-Barros R, Roque ACA.  2014.  Magnetic Aqueous Two Phase Fishing: An Hybrid Process Technology for Antibody Purification. J. Chromatogr. A. 1339:59-64. AbstractWebsite

The potential to combine aqueous two-phase extraction (ATPE) with magnetic separation was here investigated with the aim of developing a selective non-chromatographic method for the purification of antibodies from cell culture supernatants. Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and dextran were supplemented with several surface modified magnetic particles (MPs) at distinct salt concentrations. The partition of pure human IgG in the upper and lower phases as well as the amount adsorbed at the MPs surface was investigated, indicating that MPs coated with dextran and gum Arabic established the lowest amount of non-specific interactions. The binding capacity of gum arabic coated particles modified with aminophenyl boronic acid (GA-APBA-MP) was were found to be excellent in combination with the ATPS system, yielding high yields of antibody recovery (92%) and purity (98%) from cell culture supernatants. The presence of MPs in the ATPS was found to speed up phase separation (from 40 to 25 min), to consume a lower amount of MPs (half of the amount needed in magnetic fishing) and to increase the yield and purity of a mAb purified from a cell culture supernatant, when compared with ATPE or magnetic fishing processes alone.

Pina, AS, Dias AMGC, Ustok FI, Khoury GE, Fernandes CSM, Branco RJF, Lowe CR, Roque ACA.  2015.  Mild and cost-effective green fluorescent protein purification employing small synthetic ligands. Journal of Chromatography A. 1418:83-93. AbstractWebsite

Abstract The green fluorescent protein (GFP) is a useful indicator in a broad range of applications including cell biology, gene expression and biosensing. However, its full potential is hampered by the lack of a selective, mild and low-cost purification scheme. In order to address this demand, a novel adsorbent was developed as a generic platform for the purification of \{GFP\} or \{GFP\} fusion proteins, giving \{GFP\} a dual function as reporter and purification tag. After screening a solid-phase combinatorial library of small synthetic ligands based on the Ugi-reaction, the lead ligand (A4C7) selectively recovered \{GFP\} with 94% yield and 94% purity under mild conditions and directly from Escherichia coli extracts. Adsorbents containing the ligand \{A4C7\} maintained the selectivity to recover other proteins fused to GFP. The performance of \{A4C7\} adsorbents was compared with two commercially available methods (immunoprecipitation and hydrophobic interaction chromatography), confirming the new adsorbent as a low-cost viable alternative for \{GFP\} purification.

Rodrigues, R, Palma SICJ, Correia VJ, Padrao I, Pais J, Banza M, Alves C, Deuermeier J, Martins C, Costa HMA, Ramou E, Silva Pereira C, Roque ACA.  2020.  Sustainable plant polyesters as substrates for optical gas sensors. Materials Today Bio. 8:100083. AbstractPDF

The 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 stimuliresponsive 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 Xray 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.

Fernandes, CSM, Pina AS, Dias AMGC, Branco RJF, Roque ACA.  2014.  A theoretical and experimental approach toward the development of affinity adsorbents for GFP and GFP-fusion proteins purification. Journal of Biotechnology. 186:13-20. AbstractWebsite

The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena, but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward the optimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands based on a first-generation lead structure were rationally designed through molecular modeling protocols. The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugi reaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6 emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yield and purity was found for ligands A5C5 and A5C6. These were tested for purifying a model GFP-fusion protein, where ligand A5C5 yielded higher protein recovery and purity. The molecular interactions between the lead ligands and GFP were further assessed by molecular dynamics simulations, showing a wide range of potential hydrophobic and hydrogen-bond interactions.

Pina, AS, Carvalho S, Dias AMGC, Guilherme M, Pereira AS, Caraça LT, Coroadinha AS, Lowe CR, Roque ACA.  2016.  Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins. Journal of Chromatography A. 1472:55–65. AbstractWebsite

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a “tag-specific” ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11 mg ml−1 and 0.48 mg ml−1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 106 M−1 affinity constants and Qmax values of 19.11 ± 2.60 ug g−1 and 79.39 ug g−1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents.

Hussain, A, Semeano ATS, Palma SICJ, Pina AS, Almeida J, Medrado BF, Pádua ACCS, Carvalho AL, Dionísio M, Li RWC, Gamboa H, Ulijn RV, Gruber J, Roque ACA.  2017.  Tunable Gas Sensing Gels by Cooperative Assembly. Advanced Functional Materials. 1700803:1–9. AbstractPDFWebsite

The cooperative assembly of biopolymers and small molecules can yield functional materials with precisely tunable properties. Here, the fabrication, characterization, and use of multicomponent hybrid gels as selective gas sensors are reported. The gels are composed of liquid crystal droplets self-assembled in the presence of ionic liquids, which further coassemble with biopolymers to form stable matrices. Each individual component can be varied and acts cooperatively to tune gels' structure and function. The unique molecular environment in hybrid gels is explored for supramolecular recognition of volatile compounds. Gels with distinct compositions are used as optical and electrical gas sensors, yielding a combinatorial response conceptually mimicking olfactory biological systems, and tested to distinguish volatile organic compounds and to quantify ethanol in automotive fuel. The gel response is rapid, reversible, and reproducible. These robust, versatile, modular, pliant electro-optical soft materials possess new possibilities in sensing triggered by chemical and physical stimuli.

Branco, RJF, Dias AMGC, Roque ACA.  2012.  Understanding the molecular recognition between antibody fragments and protein A biomimetic ligand. Journal of Chromatography A. 1244:106-115. AbstractWebsite

Affinity chromatography with protein A from Staphylococcus aureus (SpA) is the most widespread and
accepted methodology for antibody capture during the downstream process of antibody manufacturing.
A triazine based ligand (ligand 22/8) was previously developed as an inexpensive and robust alternative
to SpA chromatography (Li et al. [12] and Teng et al. [11]). Despite the experimental success, there is no
structural information on the binding modes of ligand 22/8 to antibodies, namely to Immunoglobulin G
(IgG) molecules and fragments. In this work, we addressed this issue by a molecular docking approach
allied to molecular dynamics simulations. Theoretical results confirmed the preference of the synthetic
ligand to bind IgG through the binding site found in the crystallographic structure of the natural complex
between SpA and the Fc fragment of IgG. Our studies also suggested other unknown “hot-spots” for
specific binding of the affinity ligand at the hinge between VH and CH1 domains of Fab fragment. The best
docking poses were further analysed by molecular dynamics studies at three different protonation states
(pH 3, 7 and 11). The main interactions between ligand 22/8 and the IgG fragments found at pH 7 were
weaker at pH 3 and pH 11 and in these conditions the ligand start losing tight contact with the binding
site, corroborating the experimental evidence for protein elution from the chromatographic adsorbents
at these pH conditions.