Protein F-labeling using transglutaminase for the NMR study of intermolecular interactions.

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies.

A two-step refolding of acid-denatured microbial transglutaminase escaping from the aggregation-prone intermediate.

Microbial transglutaminase (MTG) is a monomeric globular enzyme made of 331 amino acid residues. The conformation of MTG was examined over the pH 2.0-6.

Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis.

Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca(2+)-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated.

High level expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase.

We previously observed secretion of native-type Streptomyces mobaraensis transglutaminase (MTGase) in Corynebacterium glutamicum by co-expressing the subtilisin-like protease SAM-P45 from S. albogriseolus which processes the pro-region. In the present study, we have used a chimeric pro-region consisting of S.

Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum.

We previously observed secretion of active-form transglutaminase in Corynebacterium glutamicum by coexpressing the subtilisin-like protease SAM-P45 from Streptomyces albogriseolus to process the prodomain. However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native Streptoverticillium mobaraense enzyme. In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain.

Structure of folding intermediates at pH 4.0 and native state of microbial transglutaminase.

Recombinant microbial transglutaminase has been expressed in Escherichia coli as insoluble inclusion bodies. After we searched for refolding conditions, refolding of the protein could be done by first dilution of the unfolded enzyme in a buffer at pH 4.0, and then by titration of the pH from 4.

Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus.

The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C. glutamicum.

Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense.

The crystal structure of a microbial transglutaminase from Streptoverticillium mobaraense has been determined at 2.4 A resolution. The protein folds into a plate-like shape, and has one deep cleft at the edge of the molecule.

Enhancement of transglutaminase activity by NMR identification of its flexible residues affecting the active site.

Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins, such as tofu, boiled fish paste, and sausage. By using nuclear magnetic resonance, we have shown that the residues exhibiting relatively high flexibility in MTG are localized in the N-terminal region; however, the N-terminal region influences the microenvironment of the active site. These results suggest that the N-terminal region is not of primary importance for the global fold, but influences the substrate binding.

NMR-based screening method for transglutaminases: rapid analysis of their substrate specificities and reaction rates.

Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins such as tofu, boiled fish paste, and sausage. Other transglutaminases (TGases) are expected to be used in the same way, and also to extend the scope of industrial applications to materials, drugs, and so on. The TGases have great diversity, not only in amino acid sequence and size, but also in their substrate specificities and catalytic activities, and therefore, it is quite difficult to estimate their reactivity.

Enzymatic labeling of arbitrary proteins.

This paper describes an enzymatic labeling technique (ELT), using transglutaminases. On the basis of the ELT, isotopic nuclei are easily incorporated into the gamma-carboxyamide groups of glutamine residues in arbitrary proteins, without changing their chemical structures. We have also shown that, by using ELT, protein aggregation was easily checked for NMR studies and that it can be applicable for the screening of weakly bound ligands for proteins.