title: Expression of soluble recombinant transglutaminase from Zea mays in Pichia pastoris.
abstract: Transglutaminases (TGases) catalyze post-translational protein modifications by ε-(γ-glutamyl) links and covalent amide bonds. In plant, this enzyme is poorly studied and only the Zea mays TGase gene (tgz) has been cloned. The tgz had been expressed in Escherichia coli, but the recombinant protein was mainly present in inclusion bodies. Therefore, to obtain active, soluble protein, we optimized its coding sequence according to the codon bias of Pichia pastoris and synthesized the sequence with SOEing-PCR. The optimized fragment was successfully transformed into P. pastoris GS115 by electroporation. The optimal conditions for expression were under a final concentration of 0.5 % methanol and a time-course of 96 h. The synthesized recombinant Zea mays transglutaminase (TGZs) was purified by affinity method, its production was 4.4 mg/L, and the specific activity was 0.889 U/mg under optimal expression condition. Optimal activity for TGZs was observed at 37 °C and a pH of 8.0, respectively. The cross-linking reaction of TGZs to the casein was studied, and the result was same as the reaction of casein by microbial transglutaminase. These results indicated that an effective procedure for expressing and purifying TGZs in P. pastoris GS115 was established.
authors.affiliation: School of Food Science and Engineering, Harbin Institute of Technology, Harbin 150090, China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Li
authors.forename: Lanwei
authors.initials: L
authors.lastname: Zhang
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Cui
authors.forename: Xue
authors.initials: X
authors.lastname: Luo
authors.forename: Chaohui
authors.initials: C
authors.lastname: Xue
authors.forename: Shumei
authors.initials: S
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability; D015870:Gene Expression; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D011503:Transglutaminases