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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
In ion exchange chromatography, the separation of proteins into distinct fractions occurs by selective desorption by altering the ionic strength or pH of the chromatographic medium (Nelson and Cox 2014; Procópio et al. 2017b; Patriota et al. 2017). For example, proteins strongly bound to the matrix will be eluted with a solution with greater ionic strength, while proteins that are less adsorbed will require less ionic strength to shut down. The use of saline gradients in the elution stage is very efficient in separating proteins, which—although having the same type of charge—differ in their amount. The gradient can be of the linear type, where the column is irrigated with a similar ionic strength buffer or of the ladder type, where the column is eluted with solutions with concentrations that differ in the order of 0.1 to 0.2 M (Procópio et al. 2017b; Patriota et al. 2017).
The Modification Of Carboxyl Groups
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
There are several observations on the use of l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). Figure 14 compares the rate of inactivation of yeast enolase28 with this reagent and several other carbodimides. An exogenous nucleophile was not used in these experiments. Note that l-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC) appears to be far more effective than the other two carbodiimides under these reaction conditions. In the absence of added nucleophile, the carbodiimide-activated protein carboxyl group may rearrange (Figure 15) to form a substituted O-acylurea as described by Borders and co-workers for the modification of thrombin by various water-soluble carbodiimides.29,30 The selective modification of a single aspartyl residue at the active site of lysozyme (Asp-101)31 was accomplished by the use of a low molar excess (five- to tenfold) of carbodiimide. A variety of attacking nucleophiles were used in this study (the modification reactions were performed at pH 5.0 maintained with HCl during the reaction). Of particular interest is the success achieved in the separation of the products of the reaction by ion-exchange chromatography as shown in Figure 16 and Figure 17. The modification of pancreatic phospholipase A2 (Figure 18) provides a particularly useful example of the effect of pH on carbodiimide-mediated modification. Note that the inactivation is much more rapid at pH 3.5 than at pH 5.5.
Separation Of The Bound And Unbound Forms Of The Radioactivity
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
Ion-exchange chromatography is basically a skillful game with counter-ions. One begins with a set which is already held by the fixed ions and one attempts to displace them by the ion(s) present in a solution. It has to be remembered that displacement means replacement at a 1:1 ratio. In other words, the ion displaced from the exchanger will become effective in the fluid phase. Consequently, the use of certain combinations can have detrimental effects on proteins. For example, if a protein, contained in a 1-M solution of NaCl, were to be passed through an ion exchanger with OH as the counter-ion, a hydroxyl ion would be set free for each chloride ion sorbed, and the proten would finish up in 1 M NaOH!
New generation of viral nanoparticles for targeted drug delivery in cancer therapy
Published in Journal of Drug Targeting, 2022
Nikta Alvandi, Maryam Rajabnejad, Zeynab Taghvaei, Neda Esfandiari
After VLPs assembly process, VLPs should be released from the host cells by different mechanisms which depend on VLPs structure. As shown by Figure 3(B), generally for obtaining VLPs from the culture medium or i.e. purification, three steps were recorded as follows: cell lysis and clarification, intermediate purification, and polishing. The first step initiates using 1% triton X-100 after harvesting of cell culture. Triton X-100 is a detergent utilised for cell lysis and protein extraction. Then, removing cell debris and aggregates is designed as a clarification process by centrifugation. Afterward, the intermediate purification step begins that concentration has a critical role in this process. Accordingly, adsorptive chromatography is a great choice for this step. Moreover, other chromatographic matrices based on porous membrane layers are utilised too. It should be noted that these porous matrices are devoted to large particles like VLPs. In the last step, polishing, residual host-cell protein, and DNA are removed. In this step, affinity and ion-exchange chromatography are suited. Sometimes size-exclusion chromatography is used too like when VLPs-derived impurities, such as non-assembled proteins have similar electrostatic features with VLPs. After obtaining purified VLPs, ultracentrifugation is occurred in some studies [36].
Nanoparticle-protein corona complex: understanding multiple interactions between environmental factors, corona formation, and biological activity
Published in Nanotoxicology, 2021
Aysel Tomak, Selin Cesmeli, Bercem D. Hanoglu, David Winkler, Ceyda Oksel Karakus
Differential centrifugal sedimentation (DCS) is a less commonly used particle sizing method with relatively high sensitivity. Krpetic et al. (2013) used DCS to measure the particle sizes of ligand stabilized AuNPs in the 10–50 nm range, noting its high sensitivity in detecting extremely small shell thickness variations in NPs. Similar high accuracy was achieved by Röcker et al. (2009) who successfully employed an alternative technique, fluorescence correlation spectroscopy (FCS), to quantify the adsorption of human serum albumin onto very small (<20 nm) polymer-coated NPs. Another analytical procedure traditionally used to provide information on protein fragments and aggregates is size-exclusion chromatography (SEC). This separates proteins and other molecules based on their size using their retention times. It has been successfully applied to numerous tasks, such as characterizing protein biopharmaceuticals and industrial polymers (Hong, Koza, and Bouvier 2012). It can potentially be used to determine the presence and thickness of protein corona on NPs. However, it has not been validated for use in studying structural properties of NP-protein corona complexes, due to decreased sensitivity at low concentrations of high molecular weight NP aggregates. Ion-exchange chromatography (IEC) is also frequently used for separating biomolecules of different charges. However, only a few studies involving NP-protein interactions have been reported, mainly for purifying NPs based on charge (Lévy et al. 2004).
Optimization and kinetic modeling of interchain disulfide bond reoxidation of monoclonal antibodies in bioprocesses
Published in mAbs, 2020
Peifeng Tang, Zhijun Tan, Vivekh Ehamparanathan, Tingwei Ren, Laurel Hoffman, Cheng Du, Yuanli Song, Li Tao, Angela Lewandowski, Sanchayita Ghose, Zheng Jian Li, Shijie Liu
A distinctive aspect of this study was our examination of the kinetics of disulfide formation on the Protein A resin. Here, we selected Protein A chromatography as the unit operation to implement the rescue strategy based on the following three factors. First, the affinity between mAb and Protein A ligand is through Fc region of the antibody. Since the high-order structure is intact, the affinity between mAb and Protein A would remain unchanged. Thus, reduced mAb still binds to Protein A resin. As our dynamic binding capacity study (Tan et al, mAbs, in press) showed, at 10% breakthrough and 4-min residence time, mAb material containing three different LMW levels (90%, 50%, and 1%) achieved DBCs of 58.6, 58.6, and 58.5 g/Lresin, respectively. Second, Protein A chromatography is a general unit operation to purify mAb, and implementing the rescue strategy in Protein A chromatography could be a mAb platform process operation.51,52 Third, compared with ion-exchange chromatography, Protein A chromatography is less sensitive to operation buffer composition change and easier to include the redox pair into Protein A chromatography operation buffer.53