![]() We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH‐inducible protein ligation in addition to being compatible with the widely used Sp圜atcher/Tag pair. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple‐fragment ligation. Lane 1: Novex Sharp ladder (Thermo Fisher Scientific), lane 2: SdyTag-EGFP alone, lane 3: Sd圜atcherDANG short alone, lane 4: SdyTag-EGFP incubated with Sd圜atcherDANG short, lane 5: SdyTag-EGFP incubated with Sp圜atcher, lane 6: SdyTag(Asp117Ala)-EGFP incubated with Sd圜atcherDANG short.Ī type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. (d) Covalent ligation of SdyTag-EGFP with Catchers for 80 minutes at 25☌, pH 7. Averages of triplicate measurements are shown and their standard deviations are represented by error bars. ![]() (c) Yield (%) of Catcher: SdyTag-EGFP product, with respect to the limiting Catcher substrate, from in vitro reaction of Catcher with 1.3 equivalent SdyTag-EGFP for 40 minutes at 25☌, pH 7. *Sp圜atcher sequence as follows from Zakeri et al. Residue and beta sheet numbering as follows from Fig 1B. Amino acid sequences of the constructs can be found in S1 Table. (b) Schematic diagram of the Catcher variants. Inset depicts residues at position 119 (Tag) and 34 (Catcher) for Sp圜atcher-SpyTag (pink) and Sd圜atcher-SdyTag (green). Depiction of structure was performed using PyMOL. dysgalactiae CnaB domain (green) and SpyTag-Sp圜atcher complex (pink, 4MLI ). (a) Alignment of I-TASSER homology model of S. ![]() From the success of these experiments, we foresee the application of SdyTags and SpyTags, not only, for multiplexed control of protein assembly but also for the construction of novel protein architectures. We also demonstrated that the in vivo generation of circularized proteins in a Tag-Catcher specific manner where specific Tags can be left unreacted for use in subsequent ligation reactions. These Tag-Catcher pairs were used in combination to demonstrate specific sequential assembly of tagged proteins in vitro. Similarly, SdyTag has a 75-fold specificity for its optimized Catcher, named Sd圜atcherDANG short, compared to Sp圜atcher. ![]() Sp圜atcher has 320-fold specificity for its native SpyTag compared to SdyTag. The polypeptide Tag, named SdyTag, was constructed based on the native Cna protein B-type (CnaB) domain and was found to be highly unreactive to Sp圜atcher. To extend this technology, we have engineered and characterized a new Tag-Catcher pair from a related fibronectin-binding protein in Streptococcus dysgalactiae. One of the most promising developments is a rapid protein ligation approach using a short polypeptide SpyTag and its partner, Sp圜atcher derived from Streptococcus pyogenes fibronectin-binding protein, FbaB. Over the last few years, a number of different protein assembly strategies have been developed, greatly expanding the toolbox for controlling macromolecular assembly. ![]()
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