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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
Third, the highly ingenious SplitCore technology was elaborated by the Michael Nassal’s team (Skamel et al. 2006; Nassal et al. 2008; Walker et al. 2008, 2011; Kolb et al. 2015a; Heger-Stevic et al. 2018). The SplitCore was based on the ability of two parts of the HBc molecule, coreN and coreC, to efficiently form correct HBc VLPs during expression in E. coli. The SplitCore tolerated numerous long fusions (more than 300 aa) to coreN and/or coreC and paved the way to the generation of complex triple-layered VLPs (Walker et al. 2011; Lange et al. 2014; Kolb et al. 2015a, b). The highly attractive SplitCore idea inspired other authors to present arrays of the receptor-contacting epitopes of human IgE on the surface of HBc VLPs (Baltabekova et al. 2015). The SplitCore methodology was used further to elaborate an intein-mediated trans-splicing technique, where split inteinC (intC) was added to the C-terminus of the split HBc N-core (Wang Z et al. 2021). While the split HBc with the insertion of intC at the C-terminus of N-core (designated as HBc N-intC-C) existed in inclusion bodies, the introduction of a soluble tag, gb1, to the intC C-terminus remarkably improved the solubility of recombinant protein (named HBc N-intC-gb1-C). The newly designed recombinant spontaneously assembled into the VLPs and were endowed efficiently, coupling two different model antigens onto HBc N-intC-gb1-C VLPs. The model antigens delivered by the intein-driven HBc VLP scaffold induced a dramatically enhanced antigen-specific immune response (Wang Z et al. 2021).
Hedgehog signaling in spermatogenesis and male fertility
Published in Rajender Singh, Molecular Signaling in Spermatogenesis and Male Infertility, 2019
Sandeep Kumar Bansal, Meghali Joshi, Rajender Singh
Hedgehog proteins are composed of two domains: the amino terminal “Hedge” domain (HhN) and the carboxy terminal “Hog” domain (HhC) (Figure 13.1). It was found that the carboxy-terminal domain of hh proteins is similar in sequence to the self-splicing inteins. Inteins are protein sequences that autocatalytically splice them out of the protein precursor and ligate the flanking regions into a functional protein. This region of similarity in hh proteins was named the “Hint” module.
Imaging Cellular Networks and Protein-Protein Interactions In Vivo
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
Snehal Naik, Britney L. Moss, David Piwnica-Worms, Andrea Pichler-Wallace
Most reporter complementation strategies used in optical imaging are based on either Renilla luciferase (coelenterazine substrate), or firefly and click beetle luciferases (d-luciferin substrate) (19–21). Renilla luciferases generally emit in the blue range (λmax = 475 nm) of the visible spectrum, a property less favorable for in vivo imaging, while FLucs generally emit yellow to red (λmax = 575–600 nm), enhancing their utility in vivo. Both types of luciferases have been utilized in inteinmediated protein complementation assays for detection of protein-protein interactions, developed to overcome limitations inherent to two-hybrid and fluorescence resonance energy transfer systems, such as the need for partners to be in exacting close proximity to each other or detecting interaction occurring only in the nucleus. This technique relies on posttranslational protein splicing reactions that facilitate precise excision of an intein (internal protein segment) followed by ligation of flanking exteins (external proteins) (22). The intein peptide itself is split into N- and C-terminal halves and fused in frame to each half of a reporter gene that are in turn fused in frame to protein partners of interest (23). When the two interacting proteins come together, the intein is reconstituted and spliced out, leading to reconstruction of an intact luciferase reporter gene.
Highly effective biosynthesis of N-acetylated human thymosin β4 (Tβ4) in Escherichia coli
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Rui Yu, Sai Cao, Yanhong Liu, Xinxi Si, Ting Fang, Xu Sun, Hongmei Dai, Junjie Xu, Hongqing Fang, Wei Chen
In this article, we solved two major problems in the biosynthesis of rhTβ4: N-acetylation and high expression. A fusion protein, rhTβ4-Intein, was constructed, in which rhTβ4 was fused to the N-terminus of the smallest mini-intein, Spl DnaX (from Spirulina platensis). The rhTβ4 could be fully acetylated when the rhTβ4-Intein fusion protein was expressed with the N-terminal acetyltransferase ssArd1 in E. coli. After purification and cutting by the dithiothreitol (DTT), rhTβ4 was produced with the N-terminal serine residue 100% acetylated. The yield of the N-acetylated rhTβ4 can reach more than 200 mg per litre culture fluid and about 2 grams of rhTβ4 with the purity ≥95% could be obtained after 30 L volume fermentation. The N-acetylated rhTβ4 prepared by this method showed excellent stability, activity of binding with actins from different sources and therapeutic effect on the rats with moderate to severe dry eye.
Strategies for recombinant production of antimicrobial peptides with pharmacological potential
Published in Expert Review of Clinical Pharmacology, 2020
Kamila Botelho Sampaio de Oliveira, Michel Lopes Leite, Gisele Regina Rodrigues, Harry Morales Duque, Rosiane Andrade da Costa, Victor Albuquerque Cunha, Lorena Sousa de Loiola Costa, Nicolau Brito da Cunha, Octavio Luiz Franco, Simoni Campos Dias
AMPs carrying fusion proteins use an ELP soluble expression [334]. During the purification process, ELPs aggregate with temperatures up to 30 degrees, or adding salt, while they are re-solubilized at low temperatures such as 4 degrees [329]. Nevertheless, these solubility/aggregate conditions would vary with the target protein fusion [335]. Subsequent to purification, it is necessary to release the target protein from ELP. For recombinant AMPs, intein has been used [30]. Intein is a self-cleavage system that allows the target proteins to be released [336]. AMPs such as CM4 and human β-defensin 4 were released from ELPs by self-cleavage with this system [337].
Intein mediated high throughput screening for bispecific antibodies
Published in mAbs, 2020
Tim Hofmann, Johannes Schmidt, Elke Ciesielski, Stefan Becker, Thomas Rysiok, Mark Schütte, Lars Toleikis, Harald Kolmar, Achim Doerner
In this study, we describe a novel screening methodology for bsAbs that bypasses not only chain mispairing issues, but also enables the screening of a large combinatorial space already in the desired format. The platform is based on a combinatorial approach mediated by split inteins. Split inteins are reported to be a powerful tool to avoid LC mispairing during bsAb production.[22,23] Split inteins are auto-processing domains found in nearly every organism.[24] The mechanism mediated by split inteins is called Protein Trans Splicing (PTS), and, since its discovery in 1988, it has been used for biotechnical applications, such as protein ligation,[25] purification or labeling.[26,27] In the case of ligation, a stable peptide bond is formed between two target proteins fused to their flanking exteins. Split inteins are divided into an N-terminal (IntN) and a C-terminal fragment (IntC) and remain inactive when separated from each other. After moving in close proximity, splicing occurs under soft reducing conditions undergoing several steps, as described in detail elsewhere,[28,29] resulting in a reconstituted tag-less (bispecific) antibody product. Biological functionality remains within the mild reducing environment and complex formats can be generated. For example, effector functions can be easily changed or suppressed, by switching the Fc portion. Extensive combinatorial screenings and comparison of several complex formats with comparably low production need is feasible, by applying our intein-based reconstitution approach. Antigen-binding fragment (Fab) binding moieties with different LC can be combined during screening as reconstitution occurs on the protein level, keeping their natural LCs. If needed, specific Fab engineering for retained LC pairing can be conducted subsequently and specifically focused on the identified binder only, resulting in overall faster development times.