title: Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface.
abstract: An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka, Japan. noritcm@mbox.nc.kyushu-u.ac.jp
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Satoshi
authors.initials: S
authors.lastname: Doi
authors.forename: Jo
authors.initials: J
authors.lastname: Tominaga
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Ichinose
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D002364:Caseins / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D049329:Nanostructures / Q000737:chemistry*; D010969:Plastics; D011994:Recombinant Proteins / Q000737:chemistry; D013499:Surface Properties; D011503:Transglutaminases / Q000737:chemistry*
chemical_list: D002364:Caseins; D010969:Plastics; D011994:Recombinant Proteins; D011503:Transglutaminases; D000469:Alkaline Phosphatase