Protein C-Terminal Tyrosine Conjugation via Recyclable Immobilized BmTYR

Abstract

Protein modification plays an essential role in biological and pharmaceutical research. Due to the ordinary selectivity and inevitable damage to proteins of chemical synthetic methods, increased efforts were focused on biocatalysts which exhibited high regioselectivity and mild reaction conditions. However, separation of the biocatalysts and modified proteins remained a problem, especially when scaling up. Here, we developed a simple method for site-specific protein modification with a recyclable biocatalyst. The immobilizing tyrosinase (BmTYR) on magnetic beads can oxidize C-terminal tyrosine residues of the target protein to o-quinone, followed by the spontaneous addition of different nucleophiles (e.g., aniline derivatives), resulting in a C-terminal modified protein. Compared to the homogeneous biocatalytic system reported before, this heterogeneous system leads to an easier separation. Furthermore, the solid-phase biocatalyst can be regenerated during separation, providing reusability and lower costs.

Recent developments in the cleavage, functionalization, and conjugation of proteins and peptides at tyrosine residues

“…A heterogeneous system comprising tyrosinase immobilized on magnetic beads was lately reported by Ji et al to ease the purification of protein conjugates post oxidation with the enzyme. 127 The enzyme bound to a solid phase could be readily removed from the reaction mixture by a magnetic device and was recyclable after a washing cycle with buffers. The authors then demonstrated the applicability of this reaction system in a robust peptide and protein conjugation process with aniline derivatives.…”

“…Jiang et al recently disclosed the identication of a novel 1,3,5-triazine-pyridine (TPC) reagent (127) for Tyr-specic protein conjugation (Fig. 31).…”