China 
Africa 
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Lysosomal Vitamin B12 Trafficking
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sean Froese, Matthias R. Baumgartner
DNA fragments incorporating the coding sequences of the genes of interest can be cloned in frame with N- or C-terminally tagged GFP and fRFP using established (e.g. pEGFP-N1, Clontech; pmKate2-N, Evrogen) or bespoke vectors. It is recommended that for each target protein, a GFP and an fRFP tagged construct be created because, in our experience, the nature of the protein-tag can have an effect on protein expression, even if the reasons for this are not necessarily clear. It is further important to keep in mind that GFP has severely diminished fluorescence intensity when in acidic compartments (Haupts et al., 1998), such as the lysosome, and that FRET will work poorly or not at all when fluorescent proteins are on opposite sides of the membrane. Therefore, it is recommended to use known protein structures or topologies to place the fluorescent tag on the protein terminus that is cytosolic for each target protein. In cases where protein topology is unknown, both the N- and C-termini should be separately tagged for each fluorescent protein. fRFP is recommended over shorter wavelength RFPs (e.g. DsRed or mCherry, Clontech) because the longer wavelength excitation spectrum of fRFP has essentially no overlap with the 488-nm laser, meaning very little background signal will arise from direct laser stimulation.
VLP Vaccines
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Finally, the AP205 platform was employed by the construction of the first combinatorial vaccine (Janitzek et al. 2019). Since cervical cancer and placental malaria are major public health concerns, for example, in Africa, and the target population for vaccination against both diseases, adolescent girls, would be overlapping, the authors decided to combine both vaccines by displaying the appropriate antigens on the AP205 VLPs by the plug-and-display technique. Therefore, the proof-of-concept for a combinatorial vaccine was demonstrated by simultaneous display of the two clinically relevant antigens, namely, the human papillomavirus HPV RG1 epitope and the placental malaria VAR2CSA antigen. The three distinct combinatorial VLPs were produced displaying one, two, or five concatenated RG1 epitopes without obstructing the VLP capacity to form. The co-display of VAR2CSA was achieved through a split-protein Tag/Catcher interaction without hampering the vaccine stability. Vaccination with the combinatorial vaccines was able to reduce HPV infection in vivo and induced anti-VAR2CSA IgG antibodies, which inhibited binding between native VAR2CSA expressed on infected red blood cells and chondroitin sulfate A in an in vitro binding inhibition assay (Janitzek et al. 2019). This is the first successful attempt to use the AP205 plug-and-display system to make a combinatorial vaccine capable of eliciting antibodies with dual specificity.
Emerging National and Global Nanomedicine Initiatives
Published in Harry F. Tibbals, Medical Nanotechnology and Nanomedicine, 2017
For example, the NIH-funded National Nanomedicine Center for Nucleoprotein Machines based at Georgia Tech, in collaboration with Emory University and the Medical College of Georgia, will take a biomedical engineering design approach to the repair of DNA, focusing on a model nanomachine that carries out nonhomologous end joining (NHEJ) of DNA double strand breaks. This and other DNA repair machines have relatively simple structures (<20 components) and significant biological and clinical relevance, and thus are promising as feasible models for nanoscience engineering approaches. DNA repair is vitally important to human health, as both normal metabolic activities and environmental factors can cause DNA damage, resulting in as many as 100,000 individual molecular lesions per cell per day. If allowed to accumulate without repair, these lesions interfere with gene transcription and replication, leading to premature aging, apoptosis, or unregulated cell division. The nucleoprotein machine engineering approach is as follows: Develop protein tags and fluorescence probes including quantum dot bioconjugates for nanomachine targeting.Decipher structure-function relationship for the NHEJ reaction.Characterize the dynamics of nanomachine assembly and disassembly in the repair process.Determine the dimensions and structure of repair foci at high resolution in fixed cells.Establish engineering design principles for DNA double-strand break repair.
The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Rosa Merlo, Diego Caprioglio, Michele Cillo, Anna Valenti, Rosanna Mattossovich, Castrese Morrone, Alberto Massarotti, Franca Rossi, Riccardo Miggiano, Antonio Leonardi, Alberto Minassi, Giuseppe Perugino
Concerning the specificity, we added a crude protein extract from Escherichia coli ABLE C (EcCFE), without any AGT activity at the gel-imaging analysis (Figure 4, lane 5). In this context, the only presence of the free protein-tag and the DBCO-fluorophore also did not result in any fluorescent signal (lanes 6), whereas the previously purified clickable-SNAP (lane 7), as well as its free form in the presence of BGSN3 (lane 8), was specifically able to complete the chemo-enzymatic reaction, giving an evident fluorescent signal. The high specificity of our approach was also confirmed by using the H5 enzyme, which displays a better labelling reaction than the mesophilic SNAP-tag® (Figure S6). Probably, something in the extract might impede SNAP-tag® activity. These results clearly demonstrated the high efficiency of our chemo-enzymatic approach for the labelling of both the protein-tags used.
The application of ‘kisser’ probes for resolving the distribution and microenvironment of membrane proteins in situ
Published in Journal of Neurogenetics, 2018
Michal Stawarski, Karlis Anthony Justs, Roberto Xander Hernandez, Gregory Talisker Macleod
Plasmamembrane (PM) proteins contribute to a variety of cellular processes that regulate neurodevelopment and synaptic transmission in neurons (von Heijne, 2007). PM proteins are commonly tagged to study their function and trafficking, either in live or fixed tissues, but there are shortcomings to this approach and some PM proteins (such as ion channels) cannot be easily tagged without perturbing their function. A host of tagging approaches was developed over the years, comprehensively reviewed by Crivat and Taraska (2012) and Bolbat and Schultz (2017). These include chemical tags (Jing & Cornish, 2011), peptide tags (Fritze & Anderson, 2000), fluorescent proteins (FPs), self-labeling protein tags (SNAP-tag, CLIP-tag, HALO-tag) and novel epitope tags such as Snorkel (Brown et al., 2013). As might be anticipated, there are caveats in the use of each. Generally, any translational fusion might affect folding and localization of the protein to which a peptide is appended by interfering with protein–protein interactions. Fusion proteins also disrupt cellular functions as observed in epitope-tagged ion channels (Maue, 2007). Overabundance caused by overexpression may also induce physiological changes (Shastry, 1995; Zhang et al., 2009). Although gene editing to incorporate a tag using the CRISPR/Cas9 technique can sidestep issues caused by overexpression, it remains a challenge to identify sites that will accommodate tags without disrupting protein function. Proteins, such as ion channels, have only a limited number of ‘permissive’ regions that can support protein tags without grossly affecting cell physiology (Watschinger et al., 2008).