Novel site-specific immobilization of a functional protein using a preferred substrate sequence for transglutaminase 2.
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.
Sugimura
Yoshiaki
Y
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Ueda
Hiroshi
H
Maki
Masatoshi
M
Hitomi
Kiyotaka
K
D020816:Amino Acid Motifs; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry* / Q000378:metabolism*
D000906:Antibodies; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins