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Towards the Importance of Fenugreek Proteins
Published in Dilip Ghosh, Prasad Thakurdesai, Fenugreek, 2022
Alkaline extraction/isoelectric precipitation is the most widespread protein extraction approach, and was applied for extraction of fenugreek proteins by some researchers. The technique takes advantage of high solubility of legume proteins at alkaline pH followed by their precipitation at pI (pH 4–5). This requires flour dispersion and stirring in water at appropriate ratio (usually 1:5 to 1:20) for a definite time (usually 30 to 180 min), during which the pH is adjusted at a certain amount above the isoelectric point (usually 7–11), and temperature could be elevated below the denaturation temperature (25–60 °C). Distancing from the isoelectric point increases proteins solubility. This provides higher protein concentrations in supernatant phase for precipitation at pI, which is equal to 4.5 for fenugreek proteins. This procedure could be followed by re-solubilizing of the precipitated protein at pH 7 and drying to produce final protein isolate (Boye et al., 2010a; Feyzi et al., 2015). Abdel-Aal et al (1986) extracted fenugreek proteins of Egyptian fenugreek seed, with 30.6% protein; using alkaline extraction (pH 11) followed by isoelectric precipitation the extraction reached to 60.37% protein content. Feyzi et al. (2015) applied this procedure on Iranian fenugreek seed with 34.7% protein content at various alkaline pH (6, 9, and 12), and produced fenugreek protein isolates with 90.8, 84, and 82.1% protein content, respectively. Higher protein content at lower pH might be achieved as a result of dominant contribution of albumins to fenugreek proteins (Feyzi et al., 2015).
Application of Bioresponsive Polymers in Drug Delivery
Published in Deepa H. Patel, Bioresponsive Polymers, 2020
Manisha Lalan, Deepti Jani, Pratiksha Trivedi, Deepa H. Patel
One of the platform technologies in pH-responsive systems is based on hydrogels comprised of itaconic acid copolymerized with N-vinylpyrrolidone. They were explored for the delivery of proteins exhibiting high isoelectric point. The study was carried out using salmon calcitonin and results were quite encouraging as the system showed intestinal release with faster and more extensive pH-responsive swelling than methacrylic acid-based hydrogels. Another investigation by the same group on enzymatically-degradable hydrogels of P(MAA-co-NVP) crosslinked with the peptide sequence MMRRRKK also yielded encouraging results for selective salmon calcitonin release in intestinal condition [30].
Serological Typing of HLA-A, -B, and -C Antigens
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
Isoelectric focusing (IEF) allows the definition of specificities based upon their isoelectric point. Many subtypes of HLA specificities have been identified using this method,60 but the technique’s limitation is that specificities with identical isoelectric points are indistinguishable. For example, the serologically defined specificity B703 (BPOT) has the same isoelectric point as B702, which cannot be serologically defined.32,61 Sequencing data has shown that these two specificities differ by three amino acids at position 69 to 71.62 These amino acid differences are neutral charge substitutions that explain the identical isoelectric points of these specificities. This technique is being used by some transplant centers to select the most suitable serologically matched unrelated bone marrow donors.
Construction of novel procoagulant protein targeting neuropilin-1 on tumour vasculature for tumour embolization therapy
Published in Journal of Drug Targeting, 2019
Mingyuan Zou, Malik Samiullah, Peilan Xu, Shengyu Wang, Jie He, Ting Wu, Fanghong Luo, Jianghua Yan
We used Snapgen software to analyse the open reading frame. The primary structure of the recombinant protein is shown in Figure 2(a), which is comprised of 292 amino acids encoded by 876 nucleotides. The purple fragment of 22 amino at N-terminal is the pelB signal peptide. The red fragment of 219 amino acids is tTF, and the green fragment of 27 amino acids at C-terminal is EG3287. The yellow and blue fragment is (GGGGS)3 linker and 6 × His-tag, respectively. The fundamental physiochemical properties of the recombinant protein tTF-EG3287 calculated by Expasy’s ProtParam server are shown in Table 3. The theoretical isoelectric point (pI) is 8.04, which provides reference for the pH of the protein purification buffer. The instability index is 35.29 (Instability index (II)<40 suggest the proteins as stable). Aliphatic index is a positive indicator of globular protein thermostability. The grand average of hydropathicity is –0.637, indicate recombinant protein is hydrophilic [26,27]. It is worth noting the estimated half-life of the recombinant protein tTF-EG3287 is greater than 20 h.
A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture
Published in mAbs, 2018
Chunlei Wang, Bhargavi Vemulapalli, Mingyan Cao, Dhanesh Gadre, Jihong Wang, Alan Hunter, Xiangyang Wang, Dengfeng Liu
The second step is a robust and QC-friendly method for lot release testing. The hydrophobicity difference between the correctly paired and mispaired bispecific antibodies was exploited with an HIC-based assay for GMP lot release. The capability to resolve different antibodies is a prerequisite for a generalized separation approach for mispaired bispecific antibodies. Hydrophobicity is one of the most differentiating characteristics among different monoclonal antibodies (mAbs). In fact, HIC is often employed in a high-throughput fashion to triage candidates during lead selection and developability studies. Fekete et al. have shown the wide retention time range of 17 commercial therapeutic mAbs, fusion proteins, and ADCs by using a generic fast HIC method.34 In that study, only two cases of coelution were observed. The isoelectric point is another product-specific characteristic that may be used to resolve different proteins. However, the inherent charge variants would greatly reduce the separation window, and hence limit the general application of charge-based methods for the separation of different mAbs. During our internal method evaluation, using other projects in our pipeline, we found HIC to be applicable in many more cases than the charge-based method.
Dissolution and bandgap paradigms for predicting the toxicity of metal oxide nanoparticles in the marine environment: an in vivo study with oyster embryos
Published in Nanotoxicology, 2018
Seta Noventa, Christian Hacker, Darren Rowe, Christine Elgy, Tamara Galloway
The primary physico–chemical properties of the three test NPs are summarized in Table S1 of the Supporting Information (SI). CeO2 NPs (NM-211) were obtained from the European Nanomaterial Repository (Joint Research Center, Ispra, Italy), and ZnO NPs (NM-110) from the OECD sponsorship program for the global safety assessment of nanomaterials (www.nanotechia-prospect.org). The main primary physico–chemical proprieties of these two test NPs were retrieved from the official reports (JRC64075 2011; JRC89825 2014) and from the literature (Dogra et al. 2016). The pH at the isoelectric point (pHIEP) for each NP was determined through the electrokinetic method at the NERC Facility for Environmental Nanoscience Analysis and Characterization (FENAC, Birmingham, United Kingdom).