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Order Blubervirales: Core Protein
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Seventh, insertion of the strep tag into the MIR allowed the coupling of foreign antigens, produced as streptavidin fusion proteins, on the surface of the capsids, and that allowed the use of the capsid as a universal antigen carrier (Akhras et al. 2017). Recently, the strep-tag added at the C-terminus of the HBcΔ vectors was employed for the purification needs (Aston-Deaville et al. 2020) but could be used also for the addition of desired peptides. Then, Zahn et al. (2020) fused the preS1/preS2 domain to monomeric streptavidin and produced the cargo-loaded HBc VLPs. The in vivo imaging of immunized mice demonstrated membrane permeability of cargo-loaded carriers and spread of antigens over the whole organism, which led to the destruction of the HBV-positive cells and induction of HBV-specific neutralizing antibodies. The membrane permeability of these carriers allowed for needle-free application of the antigen-loaded VLPs via oral or transdermal vaccination (Zahn et al. 2020).
Evolution
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Furthermore, the RQ135-based vectors were designed to produce a functionally active protein carrying the Strep-tag oligopeptide at its C-terminus in a standard low-cost cell-free system (Alimov et al. 2000). The presence of the Strep-tag allowed the synthesized protein to be easily isolated on a streptavidin-agarose column under mild conditions, and the entire procedure could be completed in one working day.
High-Throughput Screening for Probe Development
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Kimberly A. Kelly, Fred Reynolds, Kelly R. Kristof
Taking the HTS process of SELEX one step further, Jack Szostak has developed another method known as mRNA display (47). One of the major problems with phage display is that the bacteriophage are limiting because of the bacterial transformation requirement. With mRNA display, translation is done in vitro so that the peptide or protein can be physically linked to the nucleic acid, so genotype still equals phenotype, but the process of translation has not gone through a complex biological system. The generation of the library begins with the synthesis of mRNA oligonucleotides that terminate with puromyocin, a peptidyl acceptor and a translation-terminating antibiotic. The mRNA is translated in vitro with a commercially available kit until it comes to the puromyocin, where it enters at the A site and forms a covalent bond with the nascent peptide or protein. The conjugates isolated after mRNA display were found to bind with an affinity in the low nanomolar range, as opposed to the micromolar affinities found after phage display. For example, 20 different aptamers were found to bind to streptavidin, a commonly screened biomarker, with an affinity between 110 nM and 2.4 nM, as opposed to “strep-tag” peptide (SNWSHPQFEK) found via phage display that has a binding affinity of about 13 μΜ (48).
DMSO-tolerant ornithine decarboxylase (ODC) tandem assay optimised for high-throughput screening
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Mingu Gordon Park, Suyeon Yellena Kim, C. Justin Lee
Finally, the sample peak fractions were combined and concentrated. To evaluate the activity of the combined and concentrated human ODC, the tandem assay was performed for measuring the time-course of human ODC reaction (Figure 2F). To plot a positively sloped time-course of ODC enzyme reaction, we calculated the absolute values of Δfluorescence (|Δfluorescence|). The resulting ODC reaction plot (Figure 2G) showed that ODC enzyme reaction was saturated at 40 min, which limited the dynamic range of the assay beyond 40 min. In summary, we have successfully established the protocol for high-yield synthesis and purification of the recombinant human ODC homodimers by using human cell-line, yielding 0.7 mg of recombinant human ODC from 5 g of cell pellet for one batch. In addition, we found that Strep-tag system, not His-tag system, was suitable for purification of recombinant human ODC produced by mammalian expression system.
Improved epitope resolution of the prefusion trimer-specific antibody AM14 bound to the RSV F glycoprotein
Published in mAbs, 2021
Wayne Harshbarger, Priyanka D. Abeyrathne, Sai Tian, Ying Huang, Sumana Chandramouli, Matthew James Bottomley, Enrico Malito
Un-tagged DS-Cav1 was produced in Chinese hamster ovary cells and purified by ion exchange and size exclusion chromatography, as previously reported.26 Fab AM14 was expressed with a Strep Tag II at the heavy-chain C terminus and purified using a StrepTrap HP column (GE Healthcare). The tag was proteolytically cleaved using TEV protease (AcTEV protease, Thermo Fisher Scientific) prior to size exclusion chromatography.