Plug and play with recombinant antibody fragments

In this issue of Cell Chemical Biology, Hentrich et al., 2021Hentrich C. Kellmann S.J. Putyrski M. Cavada Hanuschka H. Knappik A. Ylera F. Periplasmic expression SpyTagged antibody fragments enables rapid modular assembly.Cell Chem. Biol. 2021; 28 (this issue): 813-824.e6Abstract Full Text PDF PubMed Scopus (5) Google Scholar describe the application SpyCatcher technology to discovery and validation. Fab-SpyTag fusion proteins can be expressed in periplasm protease-deficient bacteria coupled a manner variety SpyCatcher-tagged for improved assay performance. Various protein ligation techniques have been applied successfully engineering efforts. For example, enzymes such as sortase subtilisin (Weeks Wells, 2018Weeks A.M. Wells J.A. Engineering peptide ligase specificity by proteomic identification sites.Nat. 2018; 14: 50-57Crossref (53) Scholar, Weeks 2020Weeks Subtiligase-catalyzed ligation.Chem. Rev. 2020; 120: 3127-3160Crossref (45) Scholar) employed ligate short peptides bearing biotin or fluorescent dyes N terminus C recombinant fragments, human single-chain variable regions (scFv) Fabs. The ability label antibodies away from their antigen recognition sites ensures efficient production probes without loss functional binding. An alternative labeling strategy, involving SpyTag/SpyCatcher system, was developed fibronectin binding protein, FbaB, Streptococcus pyogenes, which 13 amino acid segment forms an isopeptide bond when it fits into groove 140 domain (Zakeri 2012Zakeri B. Fierer J.O. Celik E. Chittock E.C. Schwarz-Linek U. Moy V.T. Howarth Peptide tag forming covalent through bacterial adhesin.Proc. Natl. Acad. Sci. USA. 2012; 109: E690-E697Crossref (728) (Figure 1). This system has used extensively applications (Keeble Howarth, 2019Keeble A.H. Insider information on successful coupling with help SpyBank.Methods Enzymol. 2019; 617: 443-461Crossref (21) Scholar). versatility attracted interest its field engineering, several early reports already published. authors Fabs (Hentrich discovered that SpyTag1 fused at were subject proteolysis coli cells. (Antibody are commonly due requirement disulfide formation proper folding, is catalyzed periplasmic isomerase.) When heavy chain Fab tagged periplasm, western blots revealed His-tag C-terminal missing, whereas could detected if N-terminal SpyTag1. observation prompted research team investigate tail-specific protease (Tsp) causing SpyTag. While partial success achieved cell strain Tsp gene knocked out, group observed SpyTag2 SpyTag3, differ only few acids SpyTag1, additionally truncated proteases. As these two tags carry dibasic residues, known substrates outer membrane T (OmpT), created double-knockout (lacking OmpT activity) demonstrated excellent full-length SpyTag periplasm. With experimental solution problem hand, gone express >1,000 different carrying Flag-SpyTag2-His-tags. average yield Tsp-minus, OmpT-minus 11 mg per liter culture. then turned attention evaluating 2). anticipated, Fab-SpyTag2 form bonds soluble SpyCatcher2 10 min, evident larger molecular weight species SDS-PAGE. Dimeric oligomeric couple efficiently proteins, although longer incubation times required presumably slight impact steric hindrance. engineered unpaired cysteines allow directed chemical conjugation horseradish peroxidase (HRP) phycoerythrin (PE). Finally, SpyTag2-Fab fragment crystallizable (Fc) immunoglobulin G (IgG), called “FcCatcher,” generate bivalent, IgG-like molecule. These formats offer number benefits. First, HRP- PE-labeled SpyCatcher, secondary (i.e., goat anti-Fab-HRP, anti-Fab-PE) not background signals lower. Second, either dimeric SpyCatcher-Fc fusions, increased bivalent nature complex, permits higher apparent affinities avidity. illustrate advantages performance blots, ELISA, flow cytometry, immunofluorescence staining. approach potential streamline process characterizing validating Third, exquisite robust interaction, possible screen evaluate crude lysates hundreds cultures high-throughput manner. conclusion, convincingly establish utility assembly labeled monovalent, diverse applications. tour de force should accelerate development high-quality diagnostic purposes. will likely considered therapeutic use immunogenic (a protein), reagents easily vitro selection move forward candidates. A.K.G. C.J.M. employees Tango Biosciences. B.K.K. co-founder shareholder assemblyHentrich al.Cell BiologyFebruary 1, 2021In BriefHentrich al. report that, after knockout proteases, produced high efficiency. assembled prefabricated modules formats, reducing time effort format conversion dramatically. Full-Text Open Archive