Functional expression of secreted mouse BST-1 in yeast
Functional expression of secreted mouse BST-1 in yeast
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DOI：
10.1006/prep.1997.0811
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发表时间：
1998-02-01
影响因子：
1.6
通讯作者：
Chang, CF
中科院分区：
文献类型：
--
作者：
Hussain, AMM;Lee, HC;Chang, CF
BST-1是骨髓基质细胞表面抗原，是一种糖基磷脂酰肌醇锚定蛋白，刺激b细胞前生长，具有二磷酸腺苷(ADP)-核糖环化酶和环ADP-核糖（cADPR）水解酶活性。这两种酶活性负责cADPR的合成和水解，cADPR是一种新的钙从细胞内钙储存中释放的第二信使。人类BST-1在某些哺乳动物细胞系中的表达和鉴定已有报道。我们已经将小鼠BST-1在酵母中表达为6倍his标记的分泌蛋白。重组蛋白已被纯化，并进行了结构和功能表征。在考马斯蓝染色的SDS-PAGE凝胶上，它的表观分子质量为38.5 kDa，在Western blots上被抗BST-1的兔多克隆抗体识别。用n -聚糖酶对蛋白质进行去糖基化，产生分子量为32 - 39kda的条带梯状结构。用烟酰胺鸟嘌呤二核苷酸作为底物测定该蛋白具有adp -核糖基环化酶活性。(C) 1998学术出版社。
BST-1, a bone marrow stromal cell surface antigen, is a glycosyl phosphotidylinositol-anchored protein that stimulates pre-B-cell growth and has adenosine diphosphate (ADP)-ribosyl cyclase and cyclic ADP-ribose (cADPR) hydrolase activity. The two enzymatic activities are responsible for the synthesis and hydrolysis of cADPR, a novel second messenger of calcium release from intracellular calcium stores. The expression and characterization of human BST-1 in certain mammalian cell lines have been reported. We have expressed the murine BST-1 in yeast as a 6 x His-tagged secreted protein. The recombinant protein has been purified and subjected to structural and functional characterization. It has an apparent molecular mass of 38.5 kDa on SDS-PAGE gel stained with Coomassie blue and is recognized on Western blots by a rabbit polyclonal antibody against BST-1. Deglycosylation of the protein with N-glycanase produces a ladder of bands with molecular sizes ranging from 32 to 39 kDa. The protein possesses the ADP-ribosyl cyclase activity as measured using nicotinamide guanine dinucleotide as substrate. (C) 1998 Academic Press.