Site specific protein modification methods are crucial for their ability to confer new properties to proteins by the chemical attachment of specific functional groups. These well-defined bioconjugates can be used to study protein function or create protein-based materials. We have previously reported a protein modification strategy to site selectively modify the N-terminus of peptides and proteins using N-methylpyridinium-4-carboxaldhehyde benzene sulfonate hydrate (Rapoport's salt) as a transamination reagent, which transforms the N-terminal amino group into a uniquely reactive aldehyde or ketone. This pyruvamide functional handle can subsequently be modified in an oxime forming reaction with any alkoxyamine reagent of choice. A peptide library platform was utilized to screen for highest reactivity with Rapoport's salt by varying the three amino acids at the N-terminus. The library screen identified glutamate-glutamate-serine to be a high yielding sequence for the reaction. However, for recombinantly expressed proteins in E. coli, the presence of a glutamate residue at the first position prevents post-translational cleavage of the N-terminal methionine derived from the start codon. We will describe a way to overcome this via insertion of a proteolytic cleavage site to help yield the optimal sequence at the N-terminus. Additionally, we will present our studies on alternative high yielding sequences containing residues that can be expressed with a more facile approach. We have used site-directed mutagenesis to validate these findings on a bacterially expressed protein model, the thermophilic endoglucanase cellulase enzyme, from Pyrococcus horikoshii and determined that its enzymatic activity is not impacted by the reaction conditions, or the modification itself.

Optimization of a site selective <i>N</i>-methylpyridinium-4-carboxaldehyde mediated transamination for bacterially expressed proteins

248th National Meeting (2014)

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Optimization of a site selective N-methylpyridinium-4-carboxaldehyde mediated transamination for bacterially expressed proteins