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Tuning SpyTag-SpyCatcher mutant pairs toward orthogonal reactivity encryption.

ABSTRACT: Genetically encoded covalent peptide tagging technology, such as the SpyTag-SpyCatcher reaction, has emerged as a unique way to do chemistry with proteins. Herein, we report the reactivity engineering of SpyTag-SpyCatcher mutant pairs and show that distinct reactivity can be encrypted for the same reaction based on protein sequences of high similarity. Valuable features, including high selectivity, inverse temperature dependence and (nearly) orthogonal reactivity, could be achieved based on as few as three mutations. This demonstrates the robustness of the SpyTag-SpyCatcher reaction and the plasticity of its sequence specificity, pointing to a family of engineered protein chemistry tools.

SUBMITTER: Liu Y

PROVIDER: S-EPMC5627348 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Tuning SpyTag-SpyCatcher mutant pairs toward orthogonal reactivity encryption.

Liu Yajie Y Liu Dong D Yang Wei W Wu Xia-Ling XL Lai Luhua L Zhang Wen-Bin WB

Chemical science 20170719 9

Genetically encoded covalent peptide tagging technology, such as the SpyTag-SpyCatcher reaction, has emerged as a unique way to do chemistry with proteins. Herein, we report the reactivity engineering of SpyTag-SpyCatcher mutant pairs and show that distinct reactivity can be encrypted for the same reaction based on protein sequences of high similarity. Valuable features, including high selectivity, inverse temperature dependence and (nearly) orthogonal reactivity, could be achieved based on as f ...[more]

PMID: 28989685

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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 (SpyCatcherΔ). Target-cell-specific lentiviruses are created by mixing the Sind-SpyTag-pp and CBP-SpyCatcherΔ 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 SpyCatcherΔ 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-SpyCatcherΔ 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

Tuning SpyTag-SpyCatcher mutant pairs toward orthogonal reactivity encryption.

Publications

Tuning SpyTag-SpyCatcher mutant pairs toward orthogonal reactivity encryption.

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