Design of a Specific Peptide Tag that Affords Covalent and Site-Specific Enzyme Immobilization Catalyzed by Microbial Transglutaminase

Abstract

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)n Probe for Highly Sensitive DNA Detection

“…For the site-specific cross-linking of PfuAP and Z-QG-DNA probes using MTG, an acyl-acceptor tag sequence, MKHKGGGSGGGSGS (the underlined K is a cross-linkable site recognized by MTG) was fused to the N terminus of PfuAP (NK14-PfuAP, Figure S5 in the Supporting Information). [30] In a previous study, bacterial alkaline phosphatase (BAP) with an MKHKGS tag at the N and C termini were successfully linked to a 5'-end, Z-QG-labeled oligonucleotide by MTG at pH 8.0, [12] and the DNA-BAP conjugate retained both oligonucleotide and enzymatic function following MTG treatment. In this study, a Z-QG-DNA probe contains a number of Z-QG moieties on a strand.…”

Silica-encapsulated magnetic nanoparticles: Enzyme immobilization and cytotoxic study

“…The data reveal that the K m value of GOD-MNPs is greater than the free enzyme by a factor of about 3.3. This is probably due to high opportunities for the attachment of individual enzyme onto the surface of silica encapsulated MNPs and mass transfer limitation between substrate molecules and immobilized enzymes [5][6][7]19,[33][34][35][36]. The catalytic efficiency value, which is the ratio of k cat over K m was also different for free and immobilized enzyme.…”

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

“…The wide utility of AP in biochemical, immunological, and medical assays resulted in numerous studies investigating the immobilization of AP via fusion peptides on solid supports. In previous studies, binding of recombinant AP to various surfaces was shown via non-specific peptide tags (Tominaga et al, 2005;Zhang and Cass, 2001) and epitope conjugates of antibodies (Brennan et al, 1995). Here, we demonstrate the utility of an inorganic binding peptide as molecular linker genetically fused to bacterial AP for site-specific immobilization on the metal surface.…”