Transglutaminase Enzymatic site-selective PEGylation of proteins at lysine or glutamine

Enzymatic approaches of protein PEGylation are widening their applications thanks to the advantageous obtainment of homogeneous mono-PEGylated isomers. Microbial transglutaminase (mTGase) is an enzyme that, in nature, forms protein cross-linkings between side chains of Gln and Lys residues. For protein PEGylation, mTGase allows site-specific modification with PEG-NH2 at precise glutamines of the proteins. We have already reported mTGase-mediated conjugation with PEG-NH2 to G-CSF, yielding a site selective mono-derivative conjugate involving Gln135 [1]. Interestingly, the same enzymatic reaction of mTGase, was performed using a new PEG derivative, PEG (PEG-Z-QG), containing a Gln substrate of mTGase, in order to target the Lys residues and investigate if the selectivity of the enzyme is maintained also in this reverse conjugation method. The obtained conjugate was then compared to PEG-Q135-G-CSF isomer, in terms of biophysical characterization and pharmacokinetics [2]. In another work, an immobilized form of TGase has been studied with the aim to use it as an alternative tool for protein PEGylation that presents the advantage of a simply removal of mTGase from reaction mixture by centrifugation. The immobilized mTGase (iTGase) was characterized in term of retained activity, kinetic parameters and in different pH and temperature values. For protein PEGylation, a site-specific modification of G-CSF and α-lactalbumin (α-LA), comparing the two forms of mTGase, was then tested [3].