Covalent immobilisation of transglutaminase: stability and applications in protein PEGylation

Autor:	Antonella Grigoletto, Anna Mero, Hiroki Yoshioka, Oddone Schiavon, Gianfranco Pasut
Rok vydání:	2017
Předmět:	0301 basic medicine enzyme immobilisation PEGylation polymer conjugation protein modification Transglutaminase 3003 Tissue transglutaminase Pharmaceutical Science Peptide 02 engineering and technology Polyethylene glycol Protein Structure Secondary Polyethylene Glycols 03 medical and health sciences chemistry.chemical_compound Enzyme Stability Amino Acid Sequence chemistry.chemical_classification Chromatography Transglutaminases biology 021001 nanoscience & nanotechnology Enzymes Immobilized 030104 developmental biology Enzyme chemistry Covalent bond Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Agarose 0210 nano-technology Conjugate
Zdroj:	Journal of drug targeting. 25(9-10)
ISSN:	1029-2330
Popis:	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 Km and Vmax, respectively. Protein PEGylation was carried out using α-lactalbumin (α-LA) and granulocyte colony stimulating factor (G-CSF). In the former case, the iTGase showed a selective conjugation towards only one Gln residue of α-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.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::50cbdd808ba8a8e51f58df0be3a4a88c https://pubmed.ncbi.nlm.nih.gov/28805084 Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
Nepřihlášeným uživatelům se plný text nezobrazuje	K zobrazení výsledku je třeba se přihlásit.