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EnglishMicrobial transglutaminase enzyme (mTGase) is an extremely useful enzyme that is increasingly employed in the food and pharmaceutical industries and as a tool for protein modification and tagging. The current study describes how we immobilised mTGase (iTGase) on a solid support to improve its stability during the PEGylation process by which polyethylene glycol chains are attached to protein and peptide drugs. When the enzyme was immobilised at the N-terminal sequence on agarose beads, it retained more than 53% of its starting activity. Kinetic studies on the immobilised and free mTGase disclosed a 1.7 and 1.5 fold decrease of K-m and V-max, respectively. Protein PEGylation was carried out using alpha-lactalbumin (alpha-LA) and granulocyte colony stimulating factor (G-CSF). In the former case, the iTGase showed a selective conjugation towards only one Gln residue of alpha-LA, avoiding formation of a mono- and bi-conjugate mixture that is achieved using the free enzyme. In the latter case, the immobilised enzyme still remained selective towards only one Gln, but avoided the undesired formation of deamidated G-CSF that took place when free mTGase was used. Overall, the results of the current study highlight the suitability of iTGase in preparing site-selective protein-polymer conjugates.
| Titolo: | Covalent immobilisation of transglutaminase: stability and applications in protein PEGylation |
| Autori: | PASUT, GIANFRANCO (Corresponding) |
| Data di pubblicazione: | 2017 |
| Rivista: | |
| Abstract: | Microbial transglutaminase enzyme (mTGase) is an extremely useful enzyme that is increasingly employed in the food and pharmaceutical industries and as a tool for protein modification and tagging. The current study describes how we immobilised mTGase (iTGase) on a solid support to improve its stability during the PEGylation process by which polyethylene glycol chains are attached to protein and peptide drugs. When the enzyme was immobilised at the N-terminal sequence on agarose beads, it retained more than 53% of its starting activity. Kinetic studies on the immobilised and free mTGase disclosed a 1.7 and 1.5 fold decrease of K-m and V-max, respectively. Protein PEGylation was carried out using alpha-lactalbumin (alpha-LA) and granulocyte colony stimulating factor (G-CSF). In the former case, the iTGase showed a selective conjugation towards only one Gln residue of alpha-LA, avoiding formation of a mono- and bi-conjugate mixture that is achieved using the free enzyme. In the latter case, the immobilised enzyme still remained selective towards only one Gln, but avoided the undesired formation of deamidated G-CSF that took place when free mTGase was used. Overall, the results of the current study highlight the suitability of iTGase in preparing site-selective protein-polymer conjugates. |
| Handle: | http://hdl.handle.net/11577/3251638 |
| Appare nelle tipologie: | 01.01 - Articolo in rivista |
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