Borel F, Härtlein M, Leberman R
European Molecular Biology Laboratory, Grenoble Outstation, France.
FEBS Lett. 1993 Jun 14;324(2):162-6. doi: 10.1016/0014-5793(93)81385-d.
DNA fragments corresponding to the sequences of Escherichia coli tRNA(2ser) 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 tRNA(2ser)). From these systems 10 mg quantities of tRNA(ser)s could be isolated with a serine acceptance of 1,100 pmol/A280 unit.
与大肠杆菌tRNA(2ser)和琥珀抑制tRNA(ser)序列相对应的DNA片段，由重叠的寡核苷酸合成。这些片段被置于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。启动子、片段和终止子的基因被克隆到一个条件失控复制质粒中。在低于37摄氏度的温度下，该载体的拷贝数较低，但温度升至42摄氏度后，拷贝数不再受调控。使用这些构建体，可以使tRNA(ser)的表达量比野生型库水平高约20倍（例如相当于tRNA(2ser)表达量的200倍）。从这些系统中，可以分离出10毫克的tRNA(ser)s，其丝氨酸接受能力为1100皮摩尔/A280单位。
