TY - JOUR
T1 - An enzymatic strategy for site-specific immobilization of functional proteins using microbial transglutaminase
AU - Tominaga, Jo
AU - Kamiya, Noriho
AU - Doi, Satoshi
AU - Ichinose, Hirofumi
AU - Goto, Masahiro
N1 - Funding Information:
We are grateful to Ajinomoto Co. Inc. and Muromachi Chemical Inc. for providing MTG and polyacrylic resin samples, respectively. The present work was supported mainly by a Grant-in-Aid for Scientific Research (No. 16760638) from the Ministry of Education, Culture, Science, Sports and Technology of Japan and a grant from Kyushu Industrial Technology Center (to N.K.) and partly by the 21st Century COE Program, “Functional Innovation of Molecular Informatics” from the Ministry of Education, Culture, Science, Sports and Technology of Japan (to M.G.).
Funding Information:
MTG was provided by Ajinomoto Co. Inc. (Japan). A polyacrylic resin (PAR) (approximately 3–4 mmol carboxylic acid/g of support) was provided by Muromachi Chemical Inc. (Japan). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was purchased from Dojindo (Japan). N-Hydroxysuccinimide (NHS) was purchased from Kishida (Japan). Wild-type AP from E. coli was purchased from Wako (Japan). Bovine casein was purchased from Sigma–Aldrich (USA). The plasmid pET20-AP encoding E. coli AP gene was kindly provided by Dr. Hiroshi Ueda of the University of Tokyo. All other reagents were of commercially available analytical grade.
PY - 2004/12/1
Y1 - 2004/12/1
N2 - A novel strategy for site-specific immobilization of recombinant proteins was investigated using microbial transglutaminase (MTG). Alkaline phosphatase (AP) was selected as a model protein and tagged with a short peptide (MKHKGS) at the N-terminus to provide a reactive Lys residue for MTG. On the other hand, casein, a well-known substrate for MTG, was chemically attached onto a polyacrylic resin to provide reactive Gln residues for the enzymatic immobilization of the recombinant AP. As a result, we succeeded in MTG-mediated functional immobilization of the recombinant AP onto casein-coated polyacrylic resin. It was found that the immobilized AP prepared using MTG exhibited much higher specific activity than that prepared by chemical modification. Moreover, enzymatic immobilization gave an immobilized formulation with higher stability upon repeated use than that obtained by physical adsorption. Use of this ability of MTG in posttranslational protein modification will provide us with a benign, site-specific immobilization method for functional proteins.
AB - A novel strategy for site-specific immobilization of recombinant proteins was investigated using microbial transglutaminase (MTG). Alkaline phosphatase (AP) was selected as a model protein and tagged with a short peptide (MKHKGS) at the N-terminus to provide a reactive Lys residue for MTG. On the other hand, casein, a well-known substrate for MTG, was chemically attached onto a polyacrylic resin to provide reactive Gln residues for the enzymatic immobilization of the recombinant AP. As a result, we succeeded in MTG-mediated functional immobilization of the recombinant AP onto casein-coated polyacrylic resin. It was found that the immobilized AP prepared using MTG exhibited much higher specific activity than that prepared by chemical modification. Moreover, enzymatic immobilization gave an immobilized formulation with higher stability upon repeated use than that obtained by physical adsorption. Use of this ability of MTG in posttranslational protein modification will provide us with a benign, site-specific immobilization method for functional proteins.
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U2 - 10.1016/j.enzmictec.2004.08.014
DO - 10.1016/j.enzmictec.2004.08.014
M3 - Article
AN - SCOPUS:7544225226
SN - 0141-0229
VL - 35
SP - 613
EP - 618
JO - Enzyme and Microbial Technology
JF - Enzyme and Microbial Technology
IS - 6-7
ER -