High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme
High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme
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DOI：
10.1006/abbi.1996.0559
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发表时间：
1996-12-15
影响因子：
3.9
通讯作者：
West, CA
中科院分区：
文献类型：
--
作者：
Hill, AM;Cane, DE;West, CA
Casbene合成酶催化香叶基香叶基二磷酸（2）环化为Casbene（1），Casbene（1）是蓖麻籽（Ricinus communis L.）以应对真菌袭击。我们报道了以不溶性包涵体形式在大肠杆菌中高水平表达的cassene合酶cDNA，活性cassene合酶的增溶和重折叠，以及重组酶的动力学和产物分析。为了克服与cassene合酶基因中稀有Arg密码子的存在以及cassene本身固有的抗菌活性明显相关的问题，使用携带严格控制的T7 lac启动子的表达载体pET-21 d（+），在携带编码（t）Arg（（阿加/G））的dnaY基因的pSM 102载体的大肠杆菌宿主中表达casbene合酶基因。(C)出版社：Academic Press，Inc.
Casbene synthase catalyzes the cyclization of geranylgeranyl diphosphate (2) to casbene (1), a diterpene phytoalexin with antibacterial and antifungal activity that is produced by seedlings of castor bean (Ricinus communis L.) in response to fungal attack. We report the high-level expression of casbene synthase cDNA in Escherichia coli as insoluble inclusion bodies, the solubilization and refolding of active casbene synthase, and the kinetic and product analysis of the recombinant enzyme, To overcome problems apparently associated with the presence in the casbene synthase gene of rare Arg codons, as well as the intrinsic antibacterial activity of casbene itself, the casbene synthase gene was expressed in an E, coli host harboring the pSM102 vector that encodes the dnaY gene for (t)Arg((AGA/G)), using an expression vector, pET-21d(+), carrying the tightly controlled T7lac promoter. (C) 1996 Academic Press, Inc.