拟南芥邻氨基苯甲酸合酶亚基I在大肠杆菌中的功能表达。
Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli.
作者信息
Bernasconi P, Walters E W, Woodworth A R, Siehl D L, Stone T E, Subramanian M V
机构信息
Sandoz Agro Inc., Research Division, Palo Alto, California 94304-1104.
出版信息
Plant Physiol. 1994 Sep;106(1):353-8. doi: 10.1104/pp.106.1.353.
Abstract
Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1mg-1), the apparent K(M) for chorismate (180 microM), and the feedback inhibition by Trp (complete inhibition by 10 microM Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion project and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.
摘要
邻氨基苯甲酸合酶参与色氨酸（Trp）的生物合成。拟南芥亚基I（ASA1）在细菌中作为与谷胱甘肽S-转移酶（GST）融合的蛋白质实现了功能表达。活性产物在谷胱甘肽-琼脂糖柱上一步纯化。纯化的融合产物（GST-ASA1）的Vmax（45 nmol min-1mg-1）、对分支酸的表观K(M)（180 microM）以及色氨酸的反馈抑制（10 microM色氨酸完全抑制）与从植物中纯化的邻氨基苯甲酸合酶相当。针对融合蛋白产生的多克隆抗体，通过在GST-ASA1-琼脂糖柱上进行亲和层析纯化，与玉米幼苗部分纯化的邻氨基苯甲酸合酶制剂中的一种61.5-kD蛋白质发生交叉反应。凝血酶对GST-ASA1的切割以及色氨酸变构位点的定点诱变修饰使重组蛋白失活。
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