S-Peptide as a Potent Peptidyl Linker for Protein Cross-Linking by Microbial Transglutaminase from Streptomyces mobaraensis

Abstract

We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation.

Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

“…[61][62][63][64][65][66][67] As an extension of our previous work on an enhanced fluorescence resonance energy transfer (FRET) immunoassay, [66,67] we used SML to prepare immunoconjugates in combination with chemical and genetic techniques (Scheme 5 B). [53] Antibody fragment F-A C H T U N G T R E N N U N G (ab') 2 , prepared by the pepsin digestion of IgG, was reduced and modified with a chemically synthesized LPETG peptide (12) containing an N-terminal maleimide group.…”

Transglutaminase-catalyzed covalent multimerization of camelidae anti-human TNF single domain antibodies improves neutralizing activity

“…However, dimerization can be achieved in several other ways, e.g. chemical cross-linking, fusion of heterologous or homologous self-assembling peptides or protein domains, such as leucine zippers, helix-loop-helix motif, streptavidin and the tetramerization domain of human p53 and transglutaminases (Kamiya et al, 2003;Pluckthun and Pack, 1997;Conrath et al, 2001;Pack et al, 1995;Arndt et al, 2000;Casey et al, 1996). Oligomerization does not necessarily result in a dramatic increase in avidity of the higher ordered complex, because the independent antigen binding sites of the complex must be sterically positioned correctly to bind to separate antigen molecules simultaneously.…”

“…1A). S-tagged EGFP could be specifically cross-linked in a MTG-dependent manner (Kamiya et al, 2003). Since our aim was to posttranslationally generate bivalent and multivalent anti-hTNF-V H H antibodies, we fused the anti-hTNF-V H H cDNA with a cDNA coding for a short linker peptide and the S-tag peptide for transglutaminase mediated protein cross-linking, the c-myc-tag and his-tag coding sequences were again placed at the very N-terminus ( Fig.…”

“…Recently, MTG was used for biotinylation of monoclonal antibodies (Josten et al, 2000), and a poly(ethyleneglycol)-modification of human interleukin 2 (Sato et al, 2001). The ribonuclease S-tag-peptide was shown to work as a good peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from S. mobaraensis exemplified with S-tagged enhanced green fluorescent protein (EGFP) as a model protein (Kamiya et al, 2003).…”

Optimierung einer rekombinanten mikrobiellen Transglutaminase

“…Dies wurde bereits erfolgreich durchgeführt um GFP-Varianten (green fluorescent protein) zu verknüpfen oder wiederum Enzyme zu immobilisieren. Als Tags dienten beispielsweise Teile der F-Helix aus Myoglobin und modifizierte S-Peptid-Varianten [13] sowie kürzere Peptidsequenzen [14].…”