92 research outputs found
pH titration of ÎČ-lactoglobulin monitored by laser-based Mid-IR transmission spectroscopy coupled to chemometric analysis
A novel external cavity-quantum cascade laser (EC-QCL)-based setup for mid-IR transmission spectroscopy in the amide I and amide II region was employed for monitoring pH-induced changes of protein secondary structure. pH titration of ÎČ-lactoglobulin revealed unfolding of the native ÎČ-sheet secondary structure occurring at basic pH. Chemometric analysis of the dynamic IR spectra was performed by multivariate curve resolution-alternating least squares (MCR-ALS). Using this approach, spectral and abundance distribution profiles of the conformational transition were obtained. A proper post-processing procedure was implemented allowing to extract information about pure protein spectra and spurious signals that may interfere in the interpretation of the system. This work demonstrates the potential and versatility of the EC-QCL-based IR transmission setup for flow-through applications, benefitting from the high available optical path length.Fil: Schwaighofer, Andreas. Technische Universitat Wien; AustriaFil: Alcaraz, Mirta Raquel. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa; Argentina. Universidad Nacional del Litoral. Facultad de BioquĂmica y Ciencias BiolĂłgicas. Laboratorio de Desarrollo AnalĂtico y QuimiometrĂa; ArgentinaFil: Lux, Laurin. Technische Universitat Wien; AustriaFil: Lendl, Bernhard. Technische Universitat Wien; Austri
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Beyond Beer's Law: Why the Index of Refraction Depends (Almost) Linearly on Concentration
Beer's empiric law states that absorbance is linearly proportional to the concentration. Based on electromagnetic theory, an approximately linear dependence can only be confirmed for comparably weak oscillators. For stronger oscillators the proportionality constant, the molar attenuation coefficient, is modulated by the inverse index of refraction, which is itself a function of concentration. For comparably weak oscillators, the index of refraction function depends, like absorbance, linearly on concentration. For stronger oscillators, this linearity is lost, except at wavenumbers considerably lower than the oscillator position. In these transparency regions, linearity between the change of the index of refraction and concentration is preserved to a high degree. This can be shown with help of the KramersâKronig relations which connect the integrated absorbance to the index of refraction change at lower wavenumbers than the corresponding band. This finding builds the foundation not only for refractive index sensing, but also for new interferometric approaches in IR spectroscopy, which allow measuring the complex index of refraction function. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA
Application of Quantum Cascade Laser-Infrared Spectroscopy and Chemometrics for In-Line Discrimination of Coeluting Proteins from Preparative Size Exclusion Chromatography
An external-cavity quantum cascade laser (EC-QCL)-based flow-through mid-infrared (IR) spectrometer was placed in line with a preparative size exclusion chromatography system to demonstrate real-time analysis of protein elutions with strongly overlapping chromatographic peaks. Two different case studies involving three and four model proteins were performed under typical lab-scale purification conditions. The large optical path length (25 ÎŒm), high signal-to-noise ratios, and wide spectral coverage (1350 to 1750 cm-1) of the QCL-IR spectrometer allow for robust spectra acquisition across both the amide I and II bands. Chemometric analysis by self-modeling mixture analysis and multivariate curve resolution enabled accurate quantitation and structural fingerprinting across the protein elution transient. The acquired concentration profiles were found to be in excellent agreement with the off-line high-performance liquid chromatography reference analytics performed on the collected effluent fractions. These results demonstrate that QCL-IR detectors can be used effectively for in-line, real-time analysis of protein elutions, providing critical quality attribute data that are typically only accessible through time-consuming and resource-intensive off-line methods.Fil: Akhgar, Christopher K.. Vienna University of Technology; AustriaFil: Ebner, Julian. Vienna University of Technology; AustriaFil: Alcaraz, Mirta R.. Institute Of Chemical Technologies And Analytics; AustriaFil: Kopp, Julian. Vienna University of Technology; AustriaFil: Goicoechea, Hector Casimiro. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Santa Fe; ArgentinaFil: Spadiut, Oliver. Vienna University of Technology; AustriaFil: Schwaighofer, Andreas. Vienna University of Technology; AustriaFil: Lendl, Bernhard. Vienna University of Technology; Austri
Fine discrimination of volatile compounds by graphene-immobilized odorant-binding proteins
Abstract We describe the fabrication and performance of a biosensor for odorants, using wildtype and engineered mutants of the Italian honeybee (Apis mellifera ligustica) odorant binding protein 14 (OBP14), immobilized onto a reduced graphene oxide field-effect transistor (rGO-FET). The binding properties of the protein when immobilized on the biosensor are similar to those measured in solution, thus providing a method for measuring affinities to small molecules as an alternative to the current fluorescence assay. Out of the 14 chemicals tested, the best ligands for wildtype OBP14 were eugenol, homovanillic acid and related compounds sharing a phenol-methoxy backbone. Other chemicals, including methyl eugenol, showed affinities to OBP14 100â1000 times lower. We have also tested two mutants of OBP14. The first, bearing a HisTag at its N-terminus for better orientation on the sensor surface, showed only minor differences in its binding properties for chemicals when compared to the wildtype. The second contained an additional disulfide bond between helices α3 and α6, thus reducing the dynamics of OBP14 and leading to a higher affinity for eugenol. These data also demonstrate that it is feasible to produce biosensors with desired ligand specificities by introducing selected mutations into the structure of OBPs or other active proteins
Recombinant Protein L: Production, Purification and Characterization of a Universal Binding Ligand
Protein L (PpL) is a universal binding ligand that can be used for the detection and purification of antibodies and antibody fragments. Due to the unique interaction with immunoglobulin light chains, it differs from other affinity ligands, like protein A or G. However, due to its current higher market price, PpL is still scarce in applications. In this study, we investigated the recombinant production and purification of PpL and characterized the product in detail. We present a comprehensive roadmap for the production of the versatile protein PpL in E. coli
Teaching an old pET new tricks: tuning of inclusion body formation and properties by a mixed feed system in E. coli
Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein production, numerous biopharmaceutically important proteins are currently produced as IBs. To obtain correctly folded, soluble product, IBs have to be processed, namely, harvested, solubilized, and refolded. Several years ago, it was discovered that, depending on cultivation conditions and protein properties, IBs contain partially correctly folded protein structures, which makes IB processing more efficient. Here, we present a method of tailored induction of recombinant protein production in E. coli by a mixed feed system using glucose and lactose and its impact on IB formation. Our method allows tuning of IB amount, IB size, size distribution, and purity, which does not only facilitate IB processing, but is also crucial for potential direct applications of IBs as nanomaterials and biomaterials in regenerative medicine.COMET6676761
FTIR spectroscopy as a novel analytical approach for investigation of glucose transport and glucose transport inhibition studies in transwell in vitro barrier models
The final publication is available via https://doi.org/10.1016/j.saa.2020.118388.Glucose transport is key for cellular metabolism as well as physiological function and is maintained via passive facilitated and active sodium-glucose linked transport routes. Here, we present for the first time Fouriertransform infrared spectroscopy as a novel approach for quantification ofapical-to-basolateral glucose transport ofin vitro cell barriermodels using liver, lung, intestinal and placental cancer cell lines. Results ofour comparative study revealed that distinct differences could be observed upon subjection to transport inhibitors.European Research Counci
Validation of plasma biomarker candidates for the prediction of eGFR decline in patients with type 2 diabetes
Objective:
The decline of estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes is variable and early interventions would likely be cost effective. We elucidated the contribution of 17 plasma biomarkers to the prediction of eGFR loss on top of clinical risk factors.
Research Design and Methods:
We studied participants in PROVALID, a prospective multinational cohort study of patients with type 2 diabetes and a follow up of more than 24 months (n = 2560; baseline median eGFR 84 mL/min/1.73m2, UACR 8.1 mg/g). The 17 biomarkers were measured at baseline in 481 samples using Luminex technology and ELISA. The prediction of eGFR decline was evaluated by linear mixed modeling.
Results:
In univariable analyses nine of the 17 markers showed significant differences in median concentration between the two groups. A linear mixed model for eGFR obtained by variable selection exhibited an adjusted R2 of 62%. A panel of twelve biomarkers was selected by the procedure and accounted for 34% of the total explained variability, of which 32% were due to five markers. Each biomarkerâs individual contribution to the prediction of eGFR decline on top of clinical predictors was generally low. When included into the model, baseline eGFR exhibited the largest explained variability of eGFR decline (R2 of 79%) and the contribution of each biomarker dropped below 1%.
Conclusions:
In this longitudinal study of patients with type 2 diabetes and maintained eGFR at baseline, 12 of the 17 candidate biomarkers were associated with eGFR decline, but their predictive power was low
The impact of donor and recipient common clinical and genetic variation on estimated glomerular filtration rate in a European renal transplant population
Genetic variation across the HLA is known to influence renalâtransplant outcome. However, the impact of genetic variation beyond the HLA is less clear. We tested the association of common genetic variation and clinical characteristics, from both the donor and recipient, with postâtransplant eGFR at different timeâpoints, out to 5âyears postâtransplantation.
We conducted GWAS metaâanalyses across 10,844 donors and recipients from five European ancestry cohorts. We also analysed the impact of polygenic risk scores (PRS), calculated using genetic variants associated with nonâtransplant eGFR, on postâtransplant eGFR.
PRS calculated using the recipient genotype alone, as well as combined donor and recipient genotypes were significantly associated with eGFR at 1âyear postâtransplant. 32% of the variability in eGFR at 1âyear postâtransplant was explained by our model containing clinical covariates (including weights for death/graftâfailure), principal components and combined donorârecipient PRS, with 0.3% contributed by the PRS. No individual genetic variant was significantly associated with eGFR postâtransplant in the GWAS.
This is the first study to examine PRS, composed of variants that impact kidney function in the general population, in a postâtransplant context. Despite PRS being a significant predictor of eGFR postâtransplant, the effect size of common genetic factors is limited compared to clinical variables
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