Cloning and expression of a prokaryotic enzyme, arginine deiminase, from a primitive eukaryote Giardia intestinalis
Cloning and expression of a prokaryotic enzyme, arginine deiminase, from a primitive eukaryote Giardia intestinalis
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DOI：
10.1074/jbc.273.8.4470
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发表时间：
1998-02-20
影响因子：
4.8
通讯作者：
Edwards, MR
中科院分区：
文献类型：
--
作者：
Knodler, LA;Sekyere, EO;Edwards, MR
精氨酸脱亚胺酶（EC 3.5.3.6）催化精氨酸二氢水解酶途径中精氨酸到瓜氨酸的不可逆分解代谢。该途径被认为仅限于原核生物，但对于原始原生动物贾第鞭毛虫来说是重要的能量来源。本文报道了该虫精氨酸脱亚氨酶基因的克隆和表达。从纯化的蛋白质中，根据胰蛋白酶肽段的氨基酸序列，用简并寡核苷酸扩增精氨酸脱亚氨酶基因的一部分。然后将其用作探针以筛选HindIII和Pst I“迷你”文库，以获得含有精氨酸脱亚胺酶基因的两个重叠克隆。开放阅读框编码581个氨基酸，包括测序的所有胰蛋白酶肽，对应于67 kDa的分子量。北方印迹分析确定了一个单一的1.8-腺苷酸酶转录在滋养体和包囊细胞。精氨酸脱亚胺酶在大肠杆菌中得到了高效表达和纯化。重组蛋白被发现具有与天然酶的那些特征相媲美。
Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolisn: of arginine to citrulline in the arginine dihydrolase pathway, This pathway has been regarded as restricted to prokaryotic organisms but if an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite, Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene, The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.