Novel site-specific immobilization of a functional protein using a preferred substrate sequence for transglutaminase 2

• Published in: Journal of biotechnology 2007-08, Vol.131 (2), p.121-127
• Main Authors: Sugimura, Yoshiaki, Ueda, Hiroshi, Maki, Masatoshi, Hitomi, Kiyotaka
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Antibodies - metabolism; Binding Sites; Biological and medical sciences; Biotechnology; Cross-linking; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Fundamental and applied biological sciences. Psychology; Glutathione Transferase - chemistry; Glutathione Transferase - metabolism; Glutathione- S-transferase; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; Models, Biological; Recombinant Proteins - metabolism; Single chain fragment antibody; Substrate Specificity; Transglutaminase; Transglutaminases - chemistry; Transglutaminases - metabolism; TRANSIM
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2007.05.037

Transglutaminase (TGase) catalyzes the formation of a covalent cross-link between a peptide-bound glutamine residue and a lysine residue or primary amine. We have recently identified specific preferred sequences as glutamine-donor substrates in TGase 2 and Factor XIII reactions. By taking advantage of preference of the 12-amino acid sequence for the enzymatic reaction, an efficient immobilization method was established using two different model proteins, glutathione S-transferase (GST) and single-chain fragment antibody (scFv). Both proteins were genetically attached with the preferred substrate sequence to produce a fusion protein. Attachment of the sequence enables the recombinant proteins to act as prominent TGase-substrates and enables them to be immobilized onto chemically amine-terminated gels. Investigation of the biological activities of the two proteins demonstrated their effective immobilization in comparison with that by using a chemically immobilizing method. This established system, which we designated as Transglutaminase-mediated site-specific immobilization method (TRANSIM), would provide site-specific and biologically active conjugation between proteins and several non-protein materials.