Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library
Section snippets
Transglutaminases
The mature form of microbial transglutaminase was produced and purified from S. mobaraensis as described previously [12], [25]. Guinea pig liver TGase 2 (Sigma Chemical Co., MO) and human factor XIII (Fibrogammin RP; ZLB Behring, Marburg, Germany) were purchased. Factor XIII was activated (Factor XIIIa) by treatment with bovine thrombin (Sigma Chemical Co., MO) and then thrombin was inactivated by addition of phenylmethylsulfonyl fluoride (PMSF). In order to include as a similar enzymatic
Screening for preferred substrate sequences for MTG
A total of 1.5 × 1011 phage clones were incubated with Bio-Cd in the presence of MTG. Following the catalytic reactions, phage clones that bound Bio-Cd covalently were selected by avidin-affinity purification. These phages were subjected to four additional transamidation reaction/selection/amplification cycles. Of the total phage clones selected through five rounds of screening, 27 were randomly selected. Then, the peptide sequence displayed by each clone was determined and was aligned based on
Discussion
Initially, during the TGase catalytic reaction, the enzyme forms an intermediate with the γ-carboxyamide group of the glutamine residues in the substrate via the cysteine residue present in the active site domain. After the acyl transfer reaction to a nucleophilic substrate, such as ε-amino group of the lysine residue, this process results in the formation of intermolecluar isopeptide bond. TGase is generally more selective with regard to the participating glutamine residue in the substrate,
Acknowledgments
This work was supported by Grant-in-Aid for Scientific Research (C) No. 19580103 (to K.H.), and Grant-in-Aid for Young Scientists Research No. 186701 (to Y.S.). The first author is a research fellow at the Japan Society for the Promotion of Science.
References (31)
- et al.
Trends Biochem. Sci.
(2002) - et al.
Int. J. Biochem. Cell Biol.
(1999) - et al.
FEBS Lett.
(2005) - et al.
J. Biol. Chem.
(1998) - et al.
Protein Expr. Purif.
(2008) - et al.
J. Biol. Chem.
(1993) - et al.
J. Biotechnol.
(2005) - et al.
J. Biol. Chem.
(2006) - et al.
J. Biotechnol.
(2007) - et al.
J. Biol. Chem.
(2002)
J. Biotechnol.
J. Biosci. Bioeng.
Biochem. J.
Nat. Rev. Mol. Cell Biol.
Thromb. Haemost.
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