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Synthesis of Important Chiral Building Blocks for Pharmaceuticals Using Lactobacillus and Rhodococcus Alcohol Dehydrogenases
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Marion Rauter, Simon Krebs, Gotthard Kunze
In 2018, Döbber et al. immobilized L. brevis ADH directly from the crude cell extract by using HaloTagTM technology (Promega). Halotag-linked L. brevis ADH recombinantly expressed in E. coli was covalently bound to HaloLink Resin with high purity on a column, which was afterwards directly used for the reduction of 50 mM acetophenone to 1-(R)-phenylethanol with 95% conversion and >99% ee in a tubular reactor as shown in Fig. 13.3c. There was no activity loss found after 138 h. Although immobilized HaloTag-LbADH exhibited only a residual activity of 35% by comparison to the reference, activity losses could be compensated by organic solvents. There were only minor differences in the activity between free and immobilized enzyme in buffer/10% isopropanol with 25% and 35%. The activity was 35% for both enzyme preparations in buffer/30% THF/10% isopropanol.
Protein Degradation Inducers SNIPERs and Protacs against Oncogenic Proteins
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Norihito Shibata, Nobumichi Ohoka, Takayuki Hattori, Mikihiko Naito
SNIPERs and PROTACs require a ligand of target proteins. However, there are many proteins for which such ligands have not been found, and it is generally difficult to identify these ligands. In addition, there is no guarantee that all proteins can be degraded by protein knockdown technology, because it is dependent on various factors such as protein stability and the position of lysine residues in the proteins. To address these issues, we and others have developed a protein degrader that recognizes a tag fused to a target protein, such as the HaloTag (Neklesa et al., 2013; Tomoshige et al., 2015), hexa-His (Hattori et al., 2017; Okitsu et al., 2018), and mutated 12 kDa FK506-binding protein (FKBP12F36V) (Nabet et al., 2018). This strategy theoretically allows all target proteins to be recognized by a particular degrader without a specific ligand, and provides an easy method to validate the susceptibility of target proteins to UPS-mediated degradation by protein knockdown technology. Recently, the tag system elucidated the consequence of degrading one of the prevalent oncogenic proteins, KRAS (Nabet et al., 2018). Although the RAS family, including KRAS, is an attractive target for development of anti-tumor drugs, inhibitors targeting RAS are far from clinical translation (Cox et al., 2014). The consequence provides validation that degraders based on protein knockdown technology are a potentially new approach for RAS-targeted therapeutics, as well as the motivation to screen ligands that potently and “silently” bind to KRAS.
Advances in luminescence-based technologies for drug discovery
Published in Expert Opinion on Drug Discovery, 2023
Bolormaa Baljinnyam, Michael Ronzetti, Anton Simeonov
The development of the NLuc luciferase and its furimazine substrate led to a novel BRET platform, termed NanoBRET, created in part to address the BRET assay’s prior limitations [56]. The application of the HaloTag (HT) as an alternative for fluorescent proteins as acceptor even further advanced the NanoBRET system. HT consists of a 33 kDa protein that can be genetically fused to a protein of interest and a chloroalkane ligand that is covalently bound to the small protein. The chloroalkane ligand can bind to a range of molecules, including fluorophores [57]. This allows the use of different fluorophores in wider spectral range. NanoBRET has immensely broadened the application of BRET assays in drug discovery campaigns. The advances and applications of NanoBRET technology were recently reviewed by Dale and colleagues [58].
In silico identification and experimental validation of cellular uptake and intracellular labeling by a new cell penetrating peptide derived from CDN1
Published in Drug Delivery, 2021
Xiangli Guo, Linlin Chen, Lidan Wang, Jingping Geng, Tao Wang, Jixiong Hu, Jason Li, Changbai Liu, Hu Wang
HaloTag, a type of self-labeling protein tag, is a modified haloalkane dehalogenase derived from a bacterial enzyme. Containing 297 amino acids (33 kDa) (England et al., 2015), it cannot pass through the cell membrane without a transport vector. Data shown in this paper suggest that peptide P2 is an alternative CPP for potential macromolecule delivery. We constructed the HaloTag-P2 fusion expression plasmid, and we induced its expression and purified fusion protein via a prokaryotic protein expression system (Figure 7(A)). We also prepared protein HaloTag and HaloTag-Dot1l (a CPP found by our group (Geng et al., 2020)) as control (Figure 7(A)). Then, we followed our protocol shown in Figure 7(B) to treat HepG2 and HeLa cells. After substrate TMR incubation and washing, fluorescence images were captured. As shown in Figure 7(C,D), Figure S8(A–C), a very low amount of (0.25 µg/ml or 0.4 µg/ml protein) peptide P2 fused with HaloTag can be delivered into intracellular and cell nucleus, but the groups of TMR substrate only and HaloTag protein only do not have any signaling in the cell. These results suggest that peptide P2 not only can penetrate culture cell lines but also can deliver macromolecules like self-labeling HaloTag into cells.
Advances in cell-free protein array methods
Published in Expert Review of Proteomics, 2018
Xiaobo Yu, Brianne Petritis, Hu Duan, Danke Xu, Joshua LaBaer
Early NAPPA versions displayed GST-tagged proteins using an anti-GST antibody. In 2012, Yazaki et al. exchanged the GST fusion with Promega Corporation’s HaloTag®, a modified bacterial dehalogenase that covalently binds a small (~400 Da) chloroalkane ligand (i.e. Halo ligand). The Halo ligand is then used to capture the HaloTagged proteins instead of an antibody. HaloTag enables higher feature density since the proteins are unable to diffuse to neighboring spots once covalently immobilized to the HaloTag ligand. Using HaloTag-NAPPA, they fabricated the largest cell-free protein array of a plant proteome, containing 12,000 ORFs of the Arabidopsis thaliana plant. The proteins were expressed using a plant-based cell-free expression system derived from wheat germ extract. Protein–protein interactions for 38 transcription factors and comprehensive interactome networks were mapped using these plant-based arrays [45].