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Abstract: V zadnjih desetletjih se je biofarmacevtska industrija osredotočila na razvoj bioloških zdravil. Večinoma gre za kompleksne beljakovinske molekule, ki so zahtevne in drage za izdelavo ter imajo omejen rok uporabe, ki je posledica nestabilnosti samih beljakovin. Na nestabilnost proteinov in agregacijo lahko vplivamo z dodatkom malih molekul in pomožnih snovi kot so površinsko aktivne snovi, pufri, sladkorji, soli itd., ki se na protein neposredno vežejo in ga tako stabilizirajo. Dosedanje raziskave so že preučile vpliv adenozin tripolifosfata in tripoli fosfata na agregacijo biofarmacevtskih proteinov, zato smo v diplomskem delu preučili še vpliv petih drugih že priznanih fosfatnih ionov in za primerjavo izbrali še deset ATP- ju podobnih sulfonatnih spojin, saj so polifosfati nagnjeni k hidrolizi in posledično niso primerni za uporabo kot pomožne snovi. Uporabili smo modelni protein lizocim kokošjega beljaka ter preverjali vpliv pomožnih snovi pri različnih koncentracijah na kinetiko agregacije proteina, ki smo jo spremljali z UV- Vis spektrometrijo. S pomočjo UV-Vis spektrometrije smo opravili tudi presejalni test pomožnih snovi na agregacijo proteina in izračunali delež monomernega proteina. Ugotovili smo, da fosfatne pomožne snovi in večina sulfonatnih učinkovito zaščitijo in stabilizirajo protein in ga tako zaščitijo pred agregacijo kot je ta podvržen temperaturnemu stresu ter ga zaščitijo pred agregacijo v daljšem časovnem obdobju, prav tako tudi zmanjšajo delež monomernega proteina v vzorcu. Preverili smo tudi vpliv pomožnih snovi na agregacijo delno razvitih beljakovin, kar smo dosegli z dodatkom ditiotreitola ali DTT, ki cepi disulfidne mostičke v beljakovini. V povprečju se je izkazalo, da dajejo boljše rezultate fosfatne pomožne snovi in ne sulfonatne. Najboljše rezultate sta med pomožnimi snovmi dala ATP in TPP, preostale pomožne snovi pa dale primerljive rezultate.
Keywords: protein, agregacija, pomožne snovi, fosfati, sulfonati, fazni diagram
Published in DKUM: 01.10.2024; Views: 0; Downloads: 3
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Abstract: Rapid and reliable immunosensing is undoubtedly one of the priorities in the efficient management and combat against a pandemic, as society has experienced with the SARS-CoV-2 outbreak; simple and cost-effective sensing strategies are at the forefront of these efforts. In this regard, 2D-layered MXenes hold great potential for electrochemical biosensing due to their attractive physicochemical properties. Herein, we present a V$_2$CT$_x$ MXene-based sensing layer as an integral part of a label-free immunosensor for sensitive and selective detection of the SARS-CoV-2 spike protein. The sensor was fabricated on a supporting screen-printed carbon electrode using Nafion as an immobilizing agent for MXene and glutaraldehyde, the latter enabling effective binding of protein A for further site-oriented immobilization of anti-SARS-CoV-2 antibodies. A thorough structural analysis of the sensor architecture was carried out, and several key parameters affecting the fabrication and analytical performance of the immunosensor were investigated and optimized. The immunosensor showed excellent electroanalytical performance in combination with an impedimetric approach and exhibited a low detection limit of only 45 fM SARS-CoV-2 spike protein. Its practical applicability was successfully demonstrated by measuring the spike protein in a spiked artificial nasopharyngeal fluid sample.
Keywords: imunosenzorji, polprevodniške nanostrukture, proteini, spike protein, immunosensors, SARS-CoV-2, V2CTxMXene
Published in DKUM: 29.08.2024; Views: 42; Downloads: 1
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Abstract: The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>107 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2. A flexibly placed hydrophobic sequence motif emerges which is defined by higher order epistatic interactions between six residues, suggesting synergy that enables high connectivity in the sequence landscape. Through positive epistasis, MKK1 maintains function during mutagenesis, establishing the importance of co-dependent residues in mammalian protein kinase-substrate interactions, and creating a scenario for the evolution of diverse human signalling networks.
Keywords: protein, epistasis, catalysis, phosphate transfer, MAP kinase
Published in DKUM: 04.07.2024; Views: 104; Downloads: 7
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Keywords: resource reclamation, single cell protein, electrodialysis, forward osmosis, Saccharomyces cerevisiae
Published in DKUM: 10.05.2024; Views: 180; Downloads: 9
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Abstract: Medical implant-associated infections resulting from biofilm formation triggered by unspecific protein adsorption arethe prevailing cause of implant failure. However, implant surfaces rendered with multifunctional bioactive nanocoatings offer apromising alternative to prevent the initial attachment of bacteria and effectively interrupt biofilm formation. The need to researchand develop novel and stable bioactive nanocoatings for medical implants and a comprehensive understanding of their properties incontact with the complex biological environment are crucial. In this study, we developed an aqueous stable and crosslinker-freepolyelectrolyte−surfactant complex (PESC) composed of a renewable cationic polysaccharide, chitosan, a lysine-based anionicsurfactant (77KS), and an amphoteric antibiotic, amoxicillin, which is widely used to treat a number of infections caused by bacteria.We successfully introduced the PESC as bioactive functional nanolayers on the“model”and“real”polydimethylsiloxane (PDMS)surfaces under dynamic and ambient conditions. Besides their high stability and improved wettability, these uniformly depositednanolayers (thickness: 44−61 nm) with mixed charges exhibited strong repulsion toward three model blood proteins (serumalbumin,fibrinogen, andγ-globulin) and their competitive interactions in the mixture in real-time, as demonstrated using a quartzcrystal microbalance with dissipation (QCM-D). The functional nanolayers with a maximum negative zeta potential (ζ:−19 to−30mV at pH 7.4), water content (1628−1810 ng cm−2), and hydration (low viscosity and elastic shear modulus) correlated with themass, conformation, and interaction nature of proteins. In vitro antimicrobial activity testing under dynamic conditions showed thatthe charged nanolayers actively inhibited the growth of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcusaureus) bacteria compared to unmodified PDMS. Given the ease of fabrication of multifunctional and charged biobased coatingswith simultaneous protein-repellent and antimicrobial activities, the limitations of individual approaches could be overcome leadingto a better and advanced design of various medical devices (e.g., catheters, prosthetics, and stents).
Keywords: silicone implants, protein-repellent, antimicrobial, chitosan, lysine, bioactive coatings, adsorption, QCM-D
Published in DKUM: 15.04.2024; Views: 266; Downloads: 10
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