Transglutaminase-mediated site-specific bioconjugation of antibodies and proteins

Abstract

Non-specific chemical methods -such as acylation of amines (e.g., lysine) or alkylation of thiols (e.g., cysteine)- are widely used to construct protein conjugates for myriad of applications. These techniques have several shortcomings. First, the lack of site-specificity results in heterogenous products, as well as a synthetic route that is irreproducible and prone to side reactions. Second, modifications at or near the functional domains of proteins may significantly alter and/or reduce their activity and specificity. Conversely, site-specific methods produce homogenous, reproducible and well-defined products that are likely to achieve their desired function. A versatile and chemo-enzymatic tool to modify native (non-engineered) proteins site-specifically is using transglutaminase (TGase, E.C. 2.3.2.13). This enzyme catalyzes an iso-peptide bond between a glutaminyl amide of a protein and an amine substrate. Several methodologies were developed and their usefulness demonstrated in imaging, photoactivation and analysis. First, a facile process to build antibody-chromophore conjugates via convergent assembly was developed. This approach conferred a higher binding efficiency than its non-specific conjugation equivalent. Second, we devised a new method to modify a native antibody using TGase while retaining the antibody's primary sequence and core glycan. Previously reported methods remove the N-glycan and convert a net neutral asparagine to a negatively charged aspartic acid using PNGase F. In contrast, our new procedure not only retains the asparagine, but also the core glycan using an endoglycosidase (EndoS, E.C. 3.2.1.96). This construct is homogenous with a single glycoform and more likely to have reduced immunogenicity than the previous means. Third, a simple, robust and adaptable system was invented to generate photoremovable protein conjugates. The utility was validated by generation of a photoactivable protein, E. coli polymerase manager UmuD. These dynamic switches in protein that impart spatial and temporal control are valuable to manipulate biological systems. Lastly, an approach is presented to label glutamines with an 15N isotope in native peptides and proteins. This process obliviates the need for metabolic labeling and/or recombinant production, which are not readily accessible to many proteins and proteoforms. To the best of our knowledge, no such in-vitro labeling technique have been reported.--Author's abstract