Linking the functions of unrelated proteins using a novel directed evolution domain insertion method.
Linking the functions of unrelated proteins using a novel directed evolution domain insertion method.
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DOI：
10.1093/nar/gkn363
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发表时间：
2008-08
影响因子：
14.9
通讯作者：
Jones DD
中科院分区：
文献类型：
--
作者：
Edwards WR;Busse K;Allemann RK;Jones DD
我们已经成功地开发了一种新的定向进化方法，用于产生由一个结构域插入另一个结构域组成的完整蛋白质融合体。在插入和接受亲本结构域之间建立两个连接可以导致单独的蛋白质活动相互依赖，从而为构建分子开关提供了一种通用策略。使用工程转座子称为MuDel，从编码TEM-1 β-内酰胺酶的bla基因随机位置去除连续的三核苷酸序列。然后用编码细胞色素b562的DNA盒取代删除的三核苷酸序列，每个末端具有不同的连接序列，并采样所有三个阅读框。结果是多种嵌合基因编码新的完整融合蛋白，保留了TEM-1活性。虽然大多数耐受插入发生在环中，但也有一些发生在靠近α-螺旋和β-链末端的地方。几种变异赋予大肠杆菌切换表型，细菌对氨苄西林的耐受性依赖于生长培养基中血红素的存在。根据TEM-1内的插入位置和连接两个结构域的连接序列，切换表型的幅度从4倍到128倍不等。
We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 β-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b562 with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of α-helices and β-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.
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