Journal of Biotechnology 131 (2007) 121–127 Novel site-specific immobilization of a functional protein using a preferred substrate sequence for transglutaminase 2 Yoshiaki Sugimura a , Hiroshi Ueda b,c , Masatoshi Maki a , Kiyotaka Hitomi a,∗ a Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan b Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan c PRESTO, Japan Sciences and Technology Agency, 4-1-8, Honcho Kawaguchi, Saitama, Japan Received 2 February 2007; received in revised form 1 May 2007; accepted 25 May 2007 Abstract 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Transglutaminase; Glutathione-S-transferase; Single chain fragment antibody; Cross-linking; TRANSIM 1. Introduction Techniques for immobilization of proteins on solid sup- ports have been developed and used for various industrial and biomedical applications, including biosensors and bioreactors. The immobilization methods mainly rely on covalent attach- ment of proteins by chemical modification using aldehydes, activated esters, maleimides or epoxide cross-linkers. Although these immobilization strategies have been widely used, they generally suffer from loss of functionality due to protein denat- uration, nonspecific adsorption, and immobilization in random orientations (Cretich et al., 2006; Hultschig et al., 2006) To avoid such inactivation during the immobilization process, sev- Abbreviations: BSA, bovine serum albumin; CBB, Coomassie Bril- liant Blue; Dansyl-Cd, monodansylcadaverine; GST, glutathione S-transferase; 6xHis, hexahistidine; NHS, N-hydroxysuccinimide; OVA, ovine albumin; scFv, single chain fragment antibody; SDS, sodium dodecyl sulfate; TRANSIM, Transglutaminase-mediated site-specific immobilization method ∗ Corresponding author. Fax: +81 52 789 5542. E-mail address: hitomi@agr.nagoya-u.ac.jp (K. Hitomi). eral non-covalent or covalent approaches to enable attachment of proteins in a homogeneous orientation have been proposed. For non-covalent immobilization, several affinity tags such as hexahistidine (6xHis) and FLAG peptides (DYKDDDDK) have been employed by genetical attachment to the target pro- tein (Zhang and Cass, 2001; Wang et al., 2001). Compared to this non-covalent coupling of proteins, covalent coupling will provide continuous use due to less leakage of bound proteins. Generally, when non-covalent binding is subjected to a non- physiological condition, the ligand is liable to detach from the solid support. In respect to site-specific covalent coupling, heterobifunc- tional linker molecules have been used for cross-linking by disposing two functional groups at the either end. Such link- ers have been used for covalent coupling of a cysteine-tagged single-chain fragment antibody (scFv) to liposomes (Xu et al., 2002) and to an SPR biosensor (Torrance et al., 2006). However, application of this strategy is limited to peptide-bound cysteine residues that do not affect the function of target proteins. Enzymatic reaction is appropriate to immobilize proteins in order to overcome the defects of the chemical covalent-binding 0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.05.037