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Functional Study of Lysosomal Nutrient Transporters
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Xavier Leray, Corinne Sagné, Bruno Gasnier
Like TEVC, PC study of misrouted lysosomal transporters essentially follows classical protocols used for plasma membrane transporters. In PC recording, a single Ag/AgCl electrode records membrane voltage and injects current to clamp the membrane potential at the chosen value. One of the main differences of whole-cell PC with TEVC in oocytes is that the cytosolic compartment of the cell is dialyzed with the medium in the micropipette. Therefore, the effect of cytosolic metabolites or cytosolic protein regulators can be studied by including them in the electrode solution. This dialysis also avoids accumulating substrates within the recorded cell, which might affect transport activity over time otherwise. Heterologous expression is usually done in HEK293T cells as protein is expressed to high levels and patch-clamping can be done efficiently in this cell line.
Conversion of Natural Products from Renewable Resources in Pharmaceuticals by Cytochromes P450
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Giovanna Di Nardo, Gianfranco Gilardi
Taxol naturally occurs in the bark of the Pacific yew (Taxus brevifolia) but the concentration is very poor so that the cost is as high as $400,000 per kilogram (Li, 2015). As a consequence, the cost for dosage cycle is as much as $458, which means about $5,000 per patient affected by breast cancer. Such an issue, together with the increasing demand for this compound, has triggered intense research to synthesize this valuable compound through chemical synthesis, semi-synthesis, plant cell-culture and heterologous expression of the biosynthetic pathway in bacteria and yeast. In the last case, cytochromes P450 have been and currently are the bottleneck of the biocatalytic process. In fact, taxol synthesis from the diterpenoid precursor geranylgeranyl diphosphate involves 19 reactions with 8 of them catalyzed by cytochromes P450. The role of P450s is the hydroxylation of the terpenoid scaffold to introduce novel chiralities to the molecule that can also serve for further modifications such as glycosylation and/or the further oxidation to obtain carbonyl and carboxyl groups (Pateraki et al., 2015).
Structural Determination of the Polycystin-2 Channel by Electron Cryo-Microscopy
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Eukaryotic membrane proteins are usually harder to produce in functional form using prokaryotic expression systems, such as E. coli, because bacteria hosts lack sophisticated protein folding machinery, posttranslational modifications, and specific regulatory cellular factors, all of which are often required for maturation of these challenging membrane protein targets. For instance, membrane lipid compositions, which differ significantly between E. coli and mammalian cells, can be critical for the stability and activity of eukaryotic membrane receptors and ion channels. As a result, eukaryotic heterologous expression systems, such as insect cells and mammalian cells, are more commonly chosen to express eukaryotic membrane proteins. In particular, the BacMam system enables rapid generation of baculovirus particles in Spodoptera frugiperda insect cells, which can be subsequently used to transduce mammalian cells for protein production.83 The BacMam system avoids generation of stable cell lines, which is not only time-consuming, but can also be extremely challenging if the expression of the protein target is toxic to the host cell. Large-scale transient transfection of mammalian cells is also commonly used to express mammalian membrane proteins, but it requires preparation of a large amount of plasmids and transfection reagents that can be prohibitively expensive. Compared with the insect cell expression system, the mammalian cell expression system can be advantageous because it supports more native-like posttranslational modifications, such as glycosylation.
Methods in marine natural product drug discovery: what’s new?
Published in Expert Opinion on Drug Discovery, 2023
Jehad Almaliti, William H. Gerwick
Informatic tools are continually being developed for the efficient and targeted identification of Biosynthetic Gene Clusters (BGCs) encoding bacterial NPs of interest. For example, the ARTS tool enables the discovery of BGCs encoding for compounds with antimicrobial activity. Combination of improved tools for predicting structures from genomic information, such as antiSMASH v5, with programs that predict bioactivities from structures, are helping to sort through the enormous quantity of sequence information now available and thus focus on compounds of greatest potential [8]. Heterologous expression methodologies are now reaching a more mature stage and have thus been used to increase titer of known compounds of interest, produce compounds deriving from cryptic gene clusters or eDNA, and be engineered to produce analog substances. Another recent development has been the identification of biosynthetic gene clusters for terpene biosynthesis in the genomes of marine corals, a discovery that overturns the long-held belief that these coral-derived compounds were the product of symbiotic or otherwise associated marine microorganisms (i.e. bacteria, microalgae) [9]. The increased availability of genome sequences for other classes of eukaryotic marine organisms (e.g. sponges, tunicates) will likely drive bioinformatic propelled discoveries related to those demonstrated here with corals. These and other developments in the field are extending genome-based discovery and manipulation from the prokaryotic world into those of eukaryotes.
An oxidation resistant pediocin PA-1 derivative and penocin A display effective anti-Listeria activity in a model human gut environment
Published in Gut Microbes, 2022
Taís M. Kuniyoshi, Paula M. O’Connor, Elaine Lawton, Dinesh Thapa, Beatriz Mesa-Pereira, Sara Abulu, Colin Hill, R. Paul Ross, Ricardo P. S. Oliveira, Paul D. Cotter
Pediocin PA-1 instability due to methionine oxidation has been widely reported.20,21,29 Indeed, Johnsen et al.20 demonstrated for the first time that substituting methionine at position 31 to leucine, isoleucine or alanine prevents peptide oxidation and improves its stability. Indeed, a synthetic pediocin PA-1 analog with leucine instead of methionine (M31L) showed the same antimicrobial activity as the original peptide.20 Therefore, in this study, a pediocin M31L derivative was expressed for the first time in E. coli. This microorganism is widely used for heterologous expression due to the availability of well-established molecular tools and the ability to grow E. coli in numerous inexpensive and/or mineral media, thereby minimizing costs.30 The yield of pediocin M31L (0.725 mg/Liter of culture supernatant) obtained in this study with >95% of purity is reasonable when compared with other bacteriocin studies,19 and there is potential to further improve yield. It is worth noting that scale up of pediocin M31L production in E. coli requires further evaluation taking into account cost benefit analysis and other variables such as substrate selection. Indeed, mineral media is often used for E. coli heterologous expression and could lead to a reduction in the number of purification steps, potentially increasing yield as mature pediocin is present in the supernatant fraction.
Discovery of CFTR modulators for the treatment of cystic fibrosis
Published in Expert Opinion on Drug Discovery, 2021
Miquéias Lopes-Pacheco, Nicoletta Pedemonte, Guido Veit
In general, the development of assays for CFTR modulators based on heterologous expression systems has several advantages. First, these cells are available in large quantities, and usually their cell culture procedures are relatively easy. Second, heterologous expression systems allow to study any CFTR mutant, once the desired mutation has been introduced in the CFTR coding sequence and then, by means of an appropriate expression vector, delivered to the recipient cell. On the other hand, primary HBE cells from individuals with CF are usually available in limited number and cannot be used for large-scale studies. Primary cell cultures are usually more expensive and difficult to obtain, in particular for uncommon CFTR mutations. In addition, responsiveness of a rare CF mutation in these native cells might be difficult to assess due to the presence of a different mutation in the second allele.