Protein Conjugation via SpyStapler-Mediated SpyTag/BDTag Coupling.

Genetically encoded peptide-protein coupling reactions, such as the SpyTag/SpyCatcher chemistry, are recent additions to the expanding toolbox of protein bioconjugation. The alternative three-component ligation system, e.g., SpyStapler-mediated SpyTag/BDTag coupling, retains most advantages of the Tag/Catcher chemistry, yet requires only two short peptide tags in the genetic fusion for side-chain ligation. Not only does this facilitate the construction of large protein conjugates directly from as-expressed protein components with minimal disruption to their function, but it also provides an entirely new mode of bioconjugation via mechanical bonding, which could impart additional functional benefits such as improved activity and enhanced stability to the conjugate. Such features are attractive for improving the pharmacokinetic performance of protein therapeutics. Herein we describe protocols for SpyStapler-mediated SpyTag/BDTag coupling for protein bioconjugation. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Conjugation via isopeptide bond Support Protocol: Purification by size-exclusion chromatography Basic Protocol 2: Conjugation via mechanical bond.

An Intrinsically Disordered Peptide-Peptide Stapler for Highly Efficient Protein Ligation Both in Vivo and in Vitro.

Herein, we report an intrinsically disordered protein SpyStapler that can catalyze the isopeptide bond formation between two peptide tags, that is, SpyTag and BDTag, both in vitro and in vivo. SpyStapler and BDTag are developed by splitting SpyCatcher-the cognate protein partner of SpyTag-at the more solvent exposed second loop region. Regardless of their locations in protein constructs, SpyStapler enables efficient covalent coupling of SpyTag and BDTag under a variety of mild conditions in vitro (yield ∼80%). Co-expression of SpyStapler with telechelic dihydrofolate reductase (DHFR) bearing a SpyTag at N-terminus and a BDTag at C-terminus leads to direct cellular synthesis of a circular DHFR. Mechanistic studies involving circular dichroism and nuclear magnetic resonance spectrometry reveal that SpyStapler alone is disordered in solution and forms a stable folded structure ( Tm ∼ 55 °C) in the presence of both SpyTag and BDTag upon isopeptide bonding. No ordered structure can be formed in the absence of either tag. The catalytically inactive SpyStapler-EQ mutant cannot form a stable physical complex with SpyTag and BDTag, but it can fold into ordered structure in the presence of the ligated product (SpyTag-BDTag). It suggests that the isopeptide bond is important in stabilizing the complex. Given its efficiency, resilience, and robustness, SpyStapler provides new opportunities for bioconjugation and creation of complex protein architectures.

SpyCatcher-NTEV: A Circularly Permuted, Disordered SpyCatcher Variant for Less Trace Ligation.

The SpyTag/SpyCatcher reaction has emerged as a powerful way for bioconjugation, but it leaves a folded complex in the product after the formation of the isopeptide bond. To vary the location of the reactive residue and reduce the size of the complex and its potential immunogenicity, we engineer two circularly permuted SpyCatcher variants, SpyCatcher-N and SpyCatcher-NTEV, the latter of which possesses a TEV-recognition site for removal of the fragment containing the catalytic site. Surprisingly, both variants are found to be disordered in solution, yet still retain the ability to form an ordered complex upon reaction with SpyTag with second-order rate constants of ∼10 M-1 s-1. Cellular expression of a telechelic protein bearing SpyCatcher-NTEV at the N-terminus and SpyTag at the C-terminus gives both cyclized and chain-extended products. Notably, the monomers exist almost exclusively in the cyclic form owing to its high reactivity in vivo. The fragment containing the catalytic site of SpyCatcher-NTEV can then be removed by TEV digestion, giving a circular protein with minimal trace from the ligation reaction. The plasticity of SpyTag/SpyCatcher reactive pair has promised an ever-expanding toolbox of genetically encoded peptide-protein reaction with versatile features.

Tuning SpyTag-SpyCatcher mutant pairs toward orthogonal reactivity encryption.

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.

Topology Engineering of Proteins in Vivo Using Genetically Encoded, Mechanically Interlocking SpyX Modules for Enhanced Stability.

Recombinant proteins are traditionally limited to linear configuration. Herein, we report in vivo protein topology engineering using highly efficient, mechanically interlocking SpyX modules named AXB and BXA. SpyX modules are protein domains composed of p53dim (X), SpyTag (A), and SpyCatcher (B). The p53dim guides the intertwining of the two nascent protein chains followed by autocatalytic isopeptide bond formation between SpyTag and SpyCatcher to fulfill the interlocking, leading to a variety of backbone topologies. Direct expression of AXB or BXA produces protein catenanes with distinct ring sizes. Recombinant proteins containing SpyX modules are obtained either as mechanically interlocked obligate dimers if the protein of interest is fused to the N- or C-terminus of SpyX modules, or as star proteins if the protein is fused to both N- and C-termini. As examples, cellular syntheses of dimers of (GB1)2 (where GB1 stands for immunoglobulin-binding domain B1 of streptococcal protein G) and of four-arm elastin-like star proteins were demonstrated. Comparison of the catenation efficiencies in different constructs reveals that BXA is generally much more effective than AXB, which is rationalized by the arrangement of three domains in space. Mechanical interlocking induces considerable stability enhancement. Both AXB and BXA have a melting point ∼20 °C higher than the linear controls and the BXA catenane has a melting point ~2 °C higher than the cyclic control BX'A. Notably, four-arm elastin-like star proteins demonstrate remarkable tolerance against trypsin digestion. The SpyX modules provide a convenient and versatile approach to construct unconventional protein topologies via the "assembly-reaction" synergy, which opens a new horizon in protein science for stability enhancement and function reinforcement via topology engineering.

Phenylenediamine-based FeN(x)/C catalyst with high activity for oxygen reduction in acid medium and its active-site probing.

High-temperature pyrolyzed FeN(x)/C catalyst is one of the most promising nonprecious metal electrocatalysts for oxygen reduction reaction (ORR). However, it suffers from two challenging problems: insufficient ORR activity and unclear active site structure. Herein, we report a FeN(x)/C catalyst derived from poly-m-phenylenediamine (PmPDA-FeN(x)/C) that possesses high ORR activity (11.5 A g(-1) at 0.80 V vs RHE) and low H2O2 yield (<1%) in acid medium. The PmPDA-FeN(x)/C also exhibits high catalytic activity for both reduction and oxidation of H2O2. We further find that the ORR activity of PmPDA-FeN(x)/C is not sensitive to CO and NO(x) but can be suppressed significantly by halide ions (e.g., Cl(-), F(-), and Br(-)) and low valence state sulfur-containing species (e.g., SCN(-), SO2, and H2S). This result reveals that the active sites of the FeN(x)/C catalyst contains Fe element (mainly as Fe(III) at high potentials) in acid medium.