title: One-step purification of insoluble hydantoinase overproduced in Escherichia coli.
abstract: Over-expression of hydantoinase from Agrobacterium radiobacter NRRL B11291 (HDTar) results in the formation of insoluble aggregates in Escherichia coli. As previously reported, recombinant HDTar could be obtained in a homogeneous form using one chromatographic step. However, soluble proteins are required for the pre-treatment in several steps before proceeding to the chromatographic purification step. In this study, we reported a method based on artificial oil bodies (AOBs) to obtain homologous HDTar from its insoluble form in one step. By linkage of HDTar to intein-oleosin gene fusion, the tripartite fusion protein was over-expressed as aggregates in E. coli. Upon sonication, the mixture comprising plant oil and the insoluble fusion protein was readily assembled into AOBs. Further induction for peptide cleavage mediated by intein, the bound HDTar was liberated from AOBs, and the protein free of fusion tags was then recovered. As a result, refolded HDTar was amplified by over 300-fold. Obviously, this simplified method provides an efficient way to obtain HDTar with high yield and high purity.
authors.affiliation: Department of Life Science, National Chung-Hsing University, Taichung, Taiwan.
authors.forename: Chung-Jen
authors.initials: CJ
authors.lastname: Chiang
authors.forename: Hong-Chen
authors.initials: HC
authors.lastname: Chen
authors.forename: Yun-Peng
authors.initials: YP
authors.lastname: Chao
authors.forename: Jason T C
authors.initials: JT
authors.lastname: Tzen
mesh_terms: D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D050939:Gene Fusion; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D000581:Amidohydrolases; C020047:dihydropyrimidinase