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SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme.

ABSTRACT: SpyTag is a peptide that spontaneously forms an amide bond with its protein partner SpyCatcher. SpyTag was fused at the N terminus of ?-lactamase and SpyCatcher at the C terminus so that the partners could react to lock together the termini of the enzyme. The wild-type enzyme aggregates above 37?°C, with irreversible loss of activity. Cyclized ?-lactamase was soluble even after heating at 100?°C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher cyclization led to a much larger increase in stability than that achieved through point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding through calorimetry indicated that cyclization did not increase the unfolding temperature but rather facilitated refolding after thermal stress. SpyTag/SpyCatcher sandwiching represents a simple and efficient route to enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.

SUBMITTER: Schoene C

PROVIDER: S-EPMC4286826 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme.

Schoene Christopher C Fierer Jacob O JO Bennett S Paul SP Howarth Mark M

Angewandte Chemie (International ed. in English) 20140509 24

SpyTag is a peptide that spontaneously forms an amide bond with its protein partner SpyCatcher. SpyTag was fused at the N terminus of β-lactamase and SpyCatcher at the C terminus so that the partners could react to lock together the termini of the enzyme. The wild-type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β-lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher cyclization led to a much larg ...[more]

PMID: 24817566

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Project description:We report a simple strategy for the creation of lentiviral vectors specific to any desired target cells. SpyTag is inserted into an engineered Sindbis virus envelope protein and displayed on the lentivirus surface to create Sindbis virus-SpyTag pseudoparticles (Sind-SpyTag-pp). The SpyTag serves as the covalent anchoring site for a target-cell-specific cell-binding protein (CBP) that is fused to a truncated SpyCatcher (SpyCatcher?). Target-cell-specific lentiviruses are created by mixing the Sind-SpyTag-pp and CBP-SpyCatcher? in vitro We first used a HER2-binding designed ankyrin repeat protein (DARPin.9.26) as the model CBP. The DARPin-conjugated lentivirus transduced HER2+ SKOV3 cells with an infectious titer of 5.2 × 106 IU/ml, >500-fold higher than the unfunctionalized "naked" virions (<104 IU/ml). The ability of the DARPin-conjugated lentivirus to transduce HER2+ cells correlated with the surface expression level of HER2. Furthermore, these lentiviruses preferentially transduced HER2+ cells in cocultures containing HER2+ and HER2- cells. To enable the use of commercially available monoclonal antibodies (MAbs) as the CBP, we developed a convenient click chemistry-based approach to conjugate MAb-derived Fab fragments to a variant SpyCatcher? protein containing a nonnatural amino acid, 4-azido-l-phenylalanine (AzF). Using the HER2-binding trastuzumab as a model cell-specific MAb, we created Fab-conjugated lentiviral vectors that transduced HER2+ SKOV3 cells with an infectious titer of 2.8 × 106 IU/ml, on par with the result achieved using the DARPin-SpyCatcher? fusion protein. The ability to create cell-specific lentiviral vectors through chemical conjugation of a CBP should make this approach generalizable to any antibody, giving it broad utility for a wide range of research and clinical applications.IMPORTANCE Lentiviral vectors hold great potential in gene therapy. However, it remains a major hurdle to robustly engineer cell-specific lentiviral vectors. This article reports a simple and effective strategy to functionalize lentiviral vectors with cell-binding proteins, thus retargeting these viruses to cells expressing the binding partner of the CBP. The CBP is genetically or chemically linked to the SpyCatcher. The SpyTag is displayed on the virion surface as a fusion to an engineered Sindbis virus envelope protein and is used as the anchorage site for SpyCatcher-linked CBP. Using this strategy, we created lentiviral vectors highly infectious toward HER2+ cancer cells. The ability to rapidly create cell-specific lentiviral vectors targeting a wide range of cell types should accelerate the development of custom lentiviral vectors for many research and clinical applications.

Dataset Information

SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme.

Publications

SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme.

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