INVIVO OVEREXPRESSION AND PURIFICATION OF ESCHERICHIA-COLI TRANSFER RNA(SER)
INVIVO OVEREXPRESSION AND PURIFICATION OF ESCHERICHIA-COLI TRANSFER RNA(SER)
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DOI：
10.1016/0014-5793(93)81385-d
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发表时间：
1993-06-14
期刊：
影响因子：
3.5
通讯作者：
LEBERMAN, R
中科院分区：
文献类型：
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作者：
BOREL, F;HARTLEIN, M;LEBERMAN, R
从重叠的寡核苷酸中合成了与大肠杆菌tRNA2ser和琥珀抑制因子tRNA(Ser)序列相对应的DNA片段。它们被插入到一个强启动子和一个合成转录终止子之间，以确保产生正确大小的转录本。将启动子、片段和终止子基因克隆到条件失控复制载体中。在低于37摄氏度的温度下，该载体具有较低的拷贝数，但在温度移动到42摄氏度之后，拷贝数不再被调节。使用这些构建体，可以获得大约20倍于野生型池水平的tRNA(Ser)的过表达(对应于例如200倍于tRNA2ser的表达)。从这些系统中可以分离出10毫克量的tRNA(Serv)S，丝氨酸接受度为1,100pmol/a280)单位。
DNA fragments corresponding to the sequences of Escherichia coli tRNA2ser and amber suppressor tRNA(ser), were synthesized from overlapping oligonucleotides. These were interposed between a strong promotor and a synthetic transcriptional terminator to ensure the production of a transcript of the correct size. The genes of promotor, fragment and terminator were cloned into a conditional runaway replication plasmid. At temperatures below 37-degrees-C this vector has a low copy number but, following a temperature shift to 42-degrees-C, the copy number is no longer regulated. Using these constructs an overexpression of tRNA(ser) of about 20 times the level of the wild-type pool could be obtained (corresponding e.g. to 200 times the expression tRNA2ser). From these systems 10 mg quantities of tRNA(ser)s could be isolated with a serine acceptance of 1,100 pmol/A280) unit.