EFFICIENT EXPRESSION OF THE PARAMECIUM CALMODULIN GENE IN ESCHERICHIA-COLI AFTER 4 TAA-TO-CAA CHANGES THROUGH A SERIES OF POLYMERASE CHAIN-REACTIONS
EFFICIENT EXPRESSION OF THE PARAMECIUM CALMODULIN GENE IN ESCHERICHIA-COLI AFTER 4 TAA-TO-CAA CHANGES THROUGH A SERIES OF POLYMERASE CHAIN-REACTIONS
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DOI：
10.1111/j.1550-7408.1991.tb04814.x
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发表时间：
1991-09-01
期刊：
影响因子：
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通讯作者：
KUNG, C
中科院分区：
文献类型：
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作者：
KINK, JA;MALEY, ME;KUNG, C
我们将草履虫钙调素基因中的四个TAA密码子改为CAAs，在大肠杆菌中表达了草履虫钙调素基因。这是通过三个聚合酶链式反应（PCR）进行的，然后将产物克隆到表达载体pKK 223 -3中，紧邻其trp-lac杂合启动子的下游。JM 109株E.用含有改变的草履虫钙调蛋白基因的重组质粒转化大肠杆菌，产生被判定为钙调蛋白的蛋白。它被草履虫钙调蛋白的单克隆抗体识别;它与天然蛋白质以几乎相同的速率在草履虫中迁移;并且它在电泳模式中显示出Ca 2+依赖性的转变。钙调素的生产效率约为E.大肠杆菌与草履虫在单位体积的包装细胞，并在单位体积的液体培养效率约400倍。这种方法似乎在定点诱变和其他纤毛虫蛋白质的异源生产中是有用的。这种方法的批评提供。一个钙调素半分子，该项目的副产品，描述。
We have expressed the Paramecium calmodulin gene in Escherichia coli by changing the four TAA codons in this gene to CAAs. This was carried out by three polymerase chain reactions (PCRs) and then cloning the product into the expression vector pKK223-3 immediately downstream of its trp-lac hybrid promoter. JM109 strain of E. coli, transformed with the recombinant plasmid harboring the altered Paramecium calmodulin gene, produces a protein judged to be calmodulin. It is recognized by a monoclonal antibody to Paramecium calmodulin; it migrates with the native protein at nearly the same rate in electrophoreses; and it shows a Ca2+-dependent shift in electrophoretic pattern. The production of calmodulin is about 170 times as efficient with E. coli as with Paramecium in terms of unit volume of packed cells, and is about 400 times as efficient in unit volume of liquid culture. This method appears useful in site-directed mutageneses and in the heterologous productions of other ciliate proteins. A critique of this method is provided. A calmodulin half-molecule, a by-product of this project, is described.