Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/Sp圜atcher003. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. However, connecting at a loop is difficult because of lower flexibility and variable environment. HuCAL Fab-F-Spy2-H Negative Control Antibody 1ġ Monovalent Fab antibody with (F) DYKDDDDK-, (Spy2) SpyTag version 2, and (H) His-6-tags.There are many efficient ways to connect proteins at termini. Sp圜atcher3 fused to the hinge region, CH2, and CH3 of rabbit IgG Sp圜atcher3 fused to the hinge region, CH2, and CH3 of mouse IgG2a Sp圜atcher3 fused to the hinge region, CH2, and CH3 of human IgA Sp圜atcher3 fused to the hinge region, CH2, and CH3 of human IgG4-Pro (S228P) Sp圜atcher3 fused to the hinge region, CH2, and CH3 of human IgG4 Sp圜atcher3 fused to the hinge region, CH2, and CH3 of human IgG3 Sp圜atcher3 fused to the hinge region, CH2, and CH3 of human IgG2 Sp圜atcher3 fused to the hinge region, CH2, and CH3 of human IgG1 conjugated to Biotin Sp圜atcher3 fused to the hinge region, CH2, and CH3 of human IgG1 Sp圜atcher3 with an engineered cysteine residue use for site-specific conjugation to a label of choiceīiSp圜atcher2 with three engineered cysteine residues conjugated to biotinīiSp圜atcher2 with one engineered cysteine residue use for site-specific conjugation to a label of choiceīiSp圜atcher2 with three engineered cysteine residues use for site-specific conjugation to a label of choice Sp圜atcher2 with an engineered cysteine residue conjugated to Biotin Sp圜atcher2 with an engineered cysteine residue use for site-specific chemical conjugation to a label of choice Sp圜atchers can also be purchased from our catalog for coupling by the user to an antibody or protein with a reactive SpyTag. Sp圜atchers can be coupled to recombinant Fab antibodies as part of our TrailBlazer Antibody Custom Service. Furthermore, starting with a prelabeled Catcher enables site-directed conjugation and results in improved antibody performance.Īll Sp圜atchers and their derivatives (Table 1) are compatible with our recombinant antibodies that incorporate a SpyTag2 at the C-terminus of the heavy chain (format Fab-F-Spy2-H). We have added SpyTag to our recombinant Fab antibodies and produced a set of Sp圜atchers to 'glue' to the Fab to make TrailBlazer Antibodies.Ĭoupling an antibody with a SpyTag to different Catcher proteins increases assay design possibilities by allowing the rapid assembly of multiple, stable products, including bivalent Fabs, and Ig-like antibodies with different isotypes. The innovative SpyTag and Sp圜atcher technology, also termed 'molecular superglue', has been employed by the scientific community in many ways to build proteins with novel properties, and address the challenges of detecting and controlling proteins. Build Better Antibodies Using Molecular Superglue
0 Comments
Leave a Reply. |