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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生命科學院 > 依資料類型分類 > 期刊論文 >  One-step purification of insoluble hydantoinase overproduced in Escherichia coli

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/130741

標題: One-step purification of insoluble hydantoinase overproduced in Escherichia coli
作者: Chiang, C.J.;Chen, H.C.;Chao, Y.P.;Tzen, J.T.C.
陳鴻震;曾志正
關鍵字: hydantoinase;intein;affinity tag;artificial oil body;oleosin;artificial oil bodies;amidohydrolase;system
日期: 2007
Issue Date: 2012-12-07 16:06:39 (UTC+8)
關連: Protein Expression and Purification, Volume 52, Issue 1, Page(s) 14-18.
摘要: Over-expression of hydantoinase from Agrobacterium radiobacter NRRL B 11291 (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. (c) 2006 Elsevier Inc. All rights reserved.
Relation: Protein Expression and Purification
Appears in Collections:[依資料類型分類] 期刊論文
[依教師分類] 曾志正
[依教師分類] 陳鴻震
[依教師分類] 陳鴻震

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