Conjugation and Other Methods in Polymeric Vaccines
Mesut Karahan in Synthetic Peptide Vaccine Models, 2021
Chromatography is known as an important biophysical technique that can separate, identify, and purify the blend segments for subjective and quantitative investigation. Proteins can be purified based on properties such as size and shape, total charge, hydrophobic groups present on the surface, and the ability to bind to the stationary phase. It is based on molecular properties and interaction type use mechanism, four separation technologies, ion exchange, dispersion, surface adsorption, and size exclusion. Column chromatography is one of the most used and common techniques for protein purification methods. This technique is basically used to purify biological molecules. The application of the method can be summarized as follows. The sample is separated on the column (stationary phase) and then the wash buffer is added to the column (mobile phase). It flows through the column material placed on the fiberglass support. With the help of the wash buffer, the samples are accumulated at the bottom of the column chromatography instrument, based on time and volume (Coskun 2016). Column chromatography is a powerful purification and separation process that is closely controlled to the hydrodynamic diameters of the macromolecules depending on the diameter of the pores in the filling material (see in HPLC Method) (Acar 2006; Fornaguera and Solans 2018).
Detection of Food Allergen Residues by Immunoassays and Mass Spectrometry
Andreas L. Lopata in Food Allergy, 2017
However, one of their limitations includes detection of low abundant proteins. HPLC is the second most popular separation technique for protein purification and offers greater separation range, reproducibility and specificity. HPLC is a flexible tool with the ability to resolve large and small biomolecules with the selection of different stationary phases and offers greater sensitivity. LC coupled MS requires proper mobile phase selections devoid of salts or other ion supressing acids for successful ionisation of the analyte of interest without loss of signal and electrospray sensitivity. Protein analysis of food allergens using multidimensional protein identification technology (MudPIT) runs could provide greater separation of proteolytic peptide mixtures derived from a food sample. MudPIT is usually performed by combining two HPLC methods such as size-exclusion chromatography with reverse-phase liquid chromatography (RPLC), ion-exchange chromatography with RPLC, IEC with affinity chromatography, or affinity chromatography with RPLC. These combinations have been previously used in different food allergen studies (Fæste et al. 2011). For lC-MS/MS analysis, the amounts of starting materials, the proteolytic digestion of protein samples, and the reproducibility of identifications are critical and could affect quantification of proteins. Food allergen detection using proteomics is complicated and requires a thorough understanding and skill set to execute both instrumentation as well as downstream analysis.
Overview of Drug Development
Mark Chang, John Balser, Jim Roach, Robin Bliss in Innovative Strategies, Statistical Solutions and Simulations for Modern Clinical Trials, 2019
Following ’hits’, the lead compounds are purified using chromatographic techniques and their chemical compositions identified via spectroscopic and chemical means. Structures may be elucidated using X-ray or nuclear magnetic resonance (NMR) methods. Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is an important step in the characterization of the function, structure and interactions of the protein of interest.
Mass Spectrometry-based Biomarkers for Knee Osteoarthritis: A Systematic Review
Published in Expert Review of Proteomics, 2021
Mirella J.J. Haartmans, Kaj S. Emanuel, Gabrielle J.M. Tuijthof, Ron M. A. Heeren, Pieter J. Emans, Berta Cillero-Pastor
Post-translational modifications of proteins could also be of interest while searching for biomarkers. Top-down proteomics allows the detection of specific post-translational modifications such as phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation or acetylation, and typically involves purification steps. Glycoprotein modifications in the cartilage for instance have been described [61]. In many cases these modifications cannot be detected. Chromatographic separation or previous protein purification are often needed. In addition, protocols that disrupt collagen fibers are recommended since the access to proteins with specific roles and PTMs can be better digested then. For example, Hsueh et al. used a strong guanidine extraction buffer for protein extraction in cartilage [62].
Efficient treatment of Parkinson’s disease using ultrasonography-guided rhFGF20 proteoliposomes
Published in Drug Delivery, 2018
Jianlou Niu, Junjun Xie, Kaiwen Guo, Xiaomin Zhang, Feng Xia, Xinyu Zhao, Lintao Song, Deli Zhuge, Xiaokun Li, Yingzheng Zhao, Zhifeng Huang
The SUMO fusion system was demonstrated to enhance the soluble expression of various recombinant proteins including FGF21, FGF23, insulin-like growth factor-1 (IGF-1) (Wang et al., 2010; Sun et al., 2014; Tian et al., 2016). In the present study, we hence aimed to obtain soluble expression of bioactive rhFGF20 using the SUMO fusion tag. The pET20 expression vector harboring human FGF20 (633 bp) and a 318 bp SUMO tag was transformed into BL21 (DE3) E. coli cells (Figure 1(A)), and the expression of proteins was induced by 1 mM IPTG. The soluble and pellet fractions of the cell lysate were analyzed on a 12% (v/v) SDS–PAGE alongside other protein purification fractions (Figure 1(B)). Ni-affinity chromatography was used for the purification of SUMO-rhFGF20. And the proteins were eluted at 400 mM imidazole in 25 mM Tris–HCl buffer (pH = 8.0,150 Mm NaCl). Recombinant SUMO-rhFGF20 was identified between 35 and 40 kDa bands that corresponds to the predicted molecular mass of SUMO-rhFGF20 (Figure 1(C)).
Discovery of small molecule inhibitors of Leishmania braziliensis Hsp90 chaperone
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Fernanda A. H. Batista, Sérgio L. Ramos, Giusy Tassone, Andrei Leitão, Carlos A. Montanari, Maurizio Botta, Mattia Mori, Júlio C. Borges
The LbHsp90 and LbHsp90N (amino acid residues 1–221) recombinant proteins were expressed and purified as previously described31. Hsp90β and its N-terminal domain construct (Hsp90βN – residues 1–223) were produced as described in Minari et al.32. These plasmids were used to transform cells of Escherichia coli BL21(DE3) strain, which were grown in LB medium at 37 °C until reaching an OD600 nm about 0.6–0.8, in the presence of the appropriate antibiotic. Protein expression was induced by the addition of 0.4 mM IPTG, and kept at constant temperature for 18 h at 18 °C for hHsp90β, and 4 h at 37 °C for hHsp90βN. Induced cells were then harvested by centrifugation, and the bacterial pellet was disrupted by sonication in 20 mM sodium phosphate (pH 7.4), 20 mM imidazole, and 500 mM NaCl (20 mL of buffer/L of culture medium), after incubation with 5 U of DNAse and 30 μg/mL of lysozyme for 40 min on ice. The supernatant of the lysed cells, obtained by centrifugation at 11,000 rpm for 30 min at 4 °C, was filtered using a 0.45 μm membrane filter and subjected to protein purification protocol as described for LbHsp90 recombinant protein31. Both N-terminal constructions were incubated with 1 U of thrombin/mg of protein for 12–14 h at 4 °C for His-tag cleavage. The purification efficacy was attested by 12% SDS-PAGE. Proteins concentrations were spectroscopically determined (at 280 nm) using the molar extinction coefficient predicted by the protein amino acid sequences at water conditions.
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