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Approaches for Identification and Validation of Antimicrobial Compounds of Plant Origin: A Long Way from the Field to the Market
Published in Mahendra Rai, Chistiane M. Feitosa, Eco-Friendly Biobased Products Used in Microbial Diseases, 2022
Lívia Maria Batista Vilela, Carlos André dos Santos-Silva, Ricardo Salas Roldan-Filho, Pollyanna Michelle da Silva, Marx de Oliveira Lima, José Rafael da Silva Araújo, Wilson Dias de Oliveira, Suyane de Deus e Melo, Madson Allan de Luna Aragão, Thiago Henrique Napoleão, Patrícia Maria Guedes Paiva, Ana Christina Brasileiro-Vidal, Ana Maria Benko-Iseppon
Affinity chromatography is extremely efficient in providing a high degree of purity because it explores a specific region of the protein, that is, the specificity of a binding site (Paiva et al. 2010; Nelson and Cox 2014; Procópio et al. 2017b). It is the chromatographic process of choice if the protein of interest forms a reversible complex with a ligand. For example, a protein that contains a glucose binding site can be isolated using a column containing a glucose polymer. An enzyme can be separated from other proteins using a matrix containing substrate or inhibitor and the enzyme inhibitor will bind to a matrix that has the enzyme immobilized (Coelho et al. 2012; Pontual et al. 2014; Ferreira et al. 2019). When the affinity matrix is not commercially available, it can be produced in the laboratory by immobilizing the binder to an insoluble material such as, for example, Sepharose 4B (Silva et al. 2020). It is important to emphasize that the ligand immobilization must be via functional groups that are not involved in the interaction with the protein of interest. Also the smaller the number of regions for nonspecific binding in the matrix, the greater is its selectivity, that is, its efficiency in selecting the protein among others present in the mixture.
Receptors for Neuropeptides: Receptor Isolation Studies and Molecular Biology
Published in Edwin E. Daniel, Neuropeptide Function in the Gastrointestinal Tract, 2019
Jean-Pierre Vincent, Patrick Kitabgi
This powerful technique is the only one that can be considered as suitable for purification of membrane proteins as rare as neuropeptide receptors. Two different versions of this technique exist. In ligand affinity chromatography a ligand specifically recognized by the receptor is covalently coupled to an inert gel, whereas in immunoaffinity chromatography the specific ligand is replaced by an antibody directed against the receptor. Alternatively, when a covalent ligand-receptor complex is purified, the antibody can be specific for the ligand moiety, which remains accessible in the complex.
Separation Of The Bound And Unbound Forms Of The Radioactivity
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
Many proteins possess an affinity for a specific adsorbent, a property that can come in handy for getting rid of the unreacted radioactivity. Sometimes a simple cation-exchange chromatography (e.g., on sulfopropyl-Sephadex®) will do, as for example in the case of certain hormones,34 and a-thrombin.35,36 Affinity chromatography can be carried out in other instances with the help of small but strong columns. Thus plasminogen can be captured on Sepharose®-lysine and eluted subsequently by e-aminocaproic acid;37 antithrombin III can be adsorbed on Sepharose®-heparin and regained by salt elution.38 These are just a few examples of the temporary immobilization technique that can be applied to a number of proteins, provided the medium is inert toward the nonprotein radioactivity and the protein- bound radioactivity is fully retrievable without harsh treatment. (In this writer’s experience, insulin, for example, is not fully recoverable after adsorption on to talc.39) Adsorbing out the protein, instead of the nonprotein radioactivity, from the reaction mixture can be of advantage with regard to quality control; by applying special elution techniques (e.g., a series of steps), it is often possible to find out at an early stage whether the labeling has modified the binding properties of the protein.
In-line product quality monitoring during biopharmaceutical manufacturing using computational Raman spectroscopy
Published in mAbs, 2023
Jiarui Wang, Jingyi Chen, Joey Studts, Gang Wang
We performed several controls and regularization steps to ensure the robustness of calibration results. Affinity chromatography elution fractions provided training examples of product monomers and variants, but did not contain samples with no product or variant conditions. To prevent the prediction of protein samples under blank buffer conditions, we performed a blank affinity chromatography run without HCCF load, keeping all other variables constant. The in-line Raman spectra collected during the elution phase was then processed using the same methodology and appended to the calibration dataset. After harmonizing the training dataset with a preprocessing pipeline (see Supplementary Materials), we applied noise augmentation to reduce effects from model over-fitting and trained a panel of regression models including a convolution neural network (CNN),16 support vector regressor (SVR)17 with a nonlinear radial basis function kernel, principal component analysis regressor (PCR),25,27 and partial least squares (PLS) regressor14,19,37 (Table 2). We used a value of 2 for the number of latent variables used by the PCR and PLS models based on values used in previous studies.21 Qualitatively, the k-Nearest Neighbor (KNN) and CNN regressors were best able to predict product quality attributes that were comparable to off-line analytical results (Figure S3).
Toxoplasma gondii infection: novel emerging therapeutic targets
Published in Expert Opinion on Therapeutic Targets, 2023
Joachim Müller, Andrew Hemphill
A key step in the heuristic identification of targets of compounds issued from screening is the characterization of drug-binding proteins [48]. If we admit that a given compound uniquely or at least preferentially physically interacts with a given protein, affinity chromatography is the method of choice to identify such target proteins (Figure 2). The effective compound of interest is coupled to a suitable inert matrix. In parallel, an ineffective compound with high structural similarities to the effective compounds is coupled to the same kind of inert support. Columns filled with this material are inserted into a low-pressure liquid chromatography device. Cell-free extracts from organisms of choice are loaded into both columns in parallel. After suitable washing steps, bound proteins are eluted and identified by convenient proteomic tools. The proteomes identified in eluates from effective and ineffective compounds are compared. The differential affinoproteome, i.e. the subset of proteins binding to effective, but not to ineffective compounds, should then contain potential targets.
Surface-modified polymeric nanoparticles for drug delivery to cancer cells
Published in Expert Opinion on Drug Delivery, 2021
Arsalan Ahmed, Shumaila Sarwar, Yong Hu, Muhammad Usman Munir, Muhammad Farrukh Nisar, Fakhera Ikram, Anila Asif, Saeed Ur Rahman, Aqif Anwar Chaudhry, Ihtasham Ur Rehman
Targetability is an important characteristic in surface-modified polymeric nanoparticles. Different targeting ligands have been added on the surfaces of nanoparticles, which selectively recognize specific membrane receptors on target cells. Surface Plasmon resonance (SPR) technique is utilized to study ligand-receptor molecular association [168]. For example, Barbara and coworkers evaluated the activity of folic acid-containing nanoparticles. Folate binding protein was immobilized on the sensor surface of carboxylated dextran-coated gold film by amine coupling. The nanoparticles were allowed to interact with folate binding protein at different flow rates to study the kinetic parameters of interactions [169]. The presence or absence of targeting ligands on the surfaces of nanoparticles is also determined by affinity chromatography [170]. Bicinchoninic acid assay (BCA) and enzyme-linked immunosorbent assay (ELISA) is used if nanoparticles possess antibodies as targeting agents [171]. The Average number of targeting ligands on the surface of nanoparticles can be investigated by nuclear magnetic resonance (NMR). In this process, the integration values of signals related to targeting ligands are compared with those associated with the rest of nanoparticles [172].