Catcher/Tag Toolbox: Biomolecular Click‐Reactions For Protein Engineering Beyond Genetics

Fan, Ruxia; Aranko, A. Sesilja

doi:10.1002/cbic.202300600

Cited by 7 publications

(2 citation statements)

References 104 publications

(278 reference statements)

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“…[19] The SpyTag/SpyCatcher (ST/SC) system is a site-specific protein coupling reaction that occurs through intermolecular isopeptide binding. [20] To covalently ligate AKR with ADH, the SpyTag and SpyCatcher peptides were fused to the C-terminus or N-terminus of AKR and ADH, respectively. Unfortunately, attempting to fuse a SpyTag to the C-terminus of ADH greatly diminished its catalytic activity.…”

Section: Construction Of Fused Enzymes and Their Mutantsmentioning

confidence: 99%

Precision Engineering of the Co‐immobilization of Enzymes for Cascade Biocatalysis

Luo,

Qiao,

Chen

et al. 2024

Angew Chem Int Ed

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The design and orderly layered co‐immobilization of multiple enzymes on resin particles remain challenging. Herein, the SpyTag/SpyCatcher binding pair was fused to the N‐terminus of an alcohol dehydrogenase (ADH) and an aldo‐keto reductase (AKR), respectively. A non‐canonical amino acid (ncAA), i.e. p‐azido‐L‐phenylalanine (p‐AzF), as the anchor for covalent bonding enzymes, was genetically inserted into pre‐selected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azido‐alkynyl for the immobilization of AKR and ADH enabled the sequential dual‐enzyme coating on porous microspheres. The ordered dual‐enzyme reactor was subsequently used to asymmetrically synthesize (S)‐1‐(2‐chlorophenyl) ethanol from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74% and good reproducibility, retaining 80% of its initial activity after six cycles. The product had an enantiomeric excess (e.e.) of 99.9% that was maintained in each cycle. Additionally, the double‐layer immobilization method significantly increased the enzyme loading capacity, approximately 1.7 times greater than that of the traditional single‐layer immobilization. More importantly, it simultaneously achieves both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

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Section: Construction Of Fused Enzymes and Their Mutantsmentioning

confidence: 99%

Precision Engineering of the Co‐immobilization of Enzymes for Cascade Biocatalysis

Luo,

Qiao,

Chen

et al. 2024

Angew Chem Int Ed

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“…Despite this interaction being irreversible, the transduction of a GFP transgene in their vector was barely affected [ 298 ]. Furthermore, other versions of the tag/catcher system are pH sensitive, so they could be engineered to be biodegradable and shed after vector uptake in the endosome [ 299 ]. This system could provide another easy and attractive way of modification of Ad vectors, as the catcher molecule can be bound to a near-infinite number of antigens.…”

Section: Engineering Ads For Immune Evasionmentioning

confidence: 99%

The Immune System—A Double-Edged Sword for Adenovirus-Based Therapies

Wallace,

Bliss,

Parker

2024

Viruses

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Pathogenic adenovirus (Ad) infections are widespread but typically mild and transient, except in the immunocompromised. As vectors for gene therapy, vaccine, and oncology applications, Ad-based platforms offer advantages, including ease of genetic manipulation, scale of production, and well-established safety profiles, making them attractive tools for therapeutic development. However, the immune system often poses a significant challenge that must be overcome for adenovirus-based therapies to be truly efficacious. Both pre-existing anti-Ad immunity in the population as well as the rapid development of an immune response against engineered adenoviral vectors can have detrimental effects on the downstream impact of an adenovirus-based therapeutic. This review focuses on the different challenges posed, including pre-existing natural immunity and anti-vector immunity induced by a therapeutic, in the context of innate and adaptive immune responses. We summarise different approaches developed with the aim of tackling these problems, as well as their outcomes and potential future applications.

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Sustainable Spinning of Artificial Spider Silk Fibers with Excellent Toughness and Inherent Potential for Functionalization

Fan,

Knuuttila,

Schmuck

et al. 2024

Adv Funct Materials

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Despite impressive progress in the field, there are still several major bottlenecks in producing fibers from recombinantly produced spider‐silk‐like proteins to replicate the extraordinary mechanical properties of spider major ampullate silk. The conventional artificial fiber spinning processes rely primarily on organic solvents to coagulate proteins into fibers and require complex post‐treatments to obtain fibers with valuable properties. This is due to challenges in obtaining soluble silk proteins, but also because the native silk spinning process leading to the hierarchical organization of the silk proteins is not fully understood and is hard to replicate in a manner applicable to industrial settings. Here, recombinant spider‐silk fusion proteins are efficiently produced and processed into as‐spun fibers with a toughness modulus of 120 MJ m−3 and extensibility of 255% using solely aqueous solutions. The spider‐silk fusion proteins assemble in a manner similar to that reported for native spider silk: they phase separate induced by salting out, followed by alignment and a secondary structure transition triggered by shear forces and dehydration. Finally, the design of the fusion silk proteins enables straightforward functionalization of the fibers under mild all‐aqueous conditions via a simple biomolecular click reaction both pre‐ and post‐spinning.

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Catcher/Tag Toolbox: Biomolecular Click‐Reactions For Protein Engineering Beyond Genetics

Cited by 7 publications

References 104 publications

Precision Engineering of the Co‐immobilization of Enzymes for Cascade Biocatalysis

Precision Engineering of the Co‐immobilization of Enzymes for Cascade Biocatalysis

The Immune System—A Double-Edged Sword for Adenovirus-Based Therapies

Sustainable Spinning of Artificial Spider Silk Fibers with Excellent Toughness and Inherent Potential for Functionalization

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