Amin A A, Sadowski P D
Department of Medical Genetics, University of Toronto, Ontario, Canada.
Mol Cell Biol. 1989 May;9(5):1987-95. doi: 10.1128/mcb.9.5.1987-1995.1989.
We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.
我们使用了一种体外转录和翻译系统来合成具有酶活性的FLP蛋白。由SP6聚合酶在体外合成的FLP mRNA在兔网织红细胞裂解物中能高效翻译，从而产生具有酶活性的FLP。利用该系统，我们评估了缺失和四肽插入对体外合成的各个变体蛋白与FLP识别靶位点结合以及进行切除重组能力的影响。从FLP的羧基末端或氨基末端区域删除少至六个氨基酸会导致结合活性丧失。同样，在氨基酸位置79、203和286处的插入消除了DNA结合活性。另一方面，在氨基酸364处有插入的蛋白保留了显著的DNA结合活性，但没有可检测到的重组活性。此外，在氨基酸115处的插入对DNA结合没有可测量的影响，但重组减少了95%。另外，在氨基酸411处的插入对DNA结合和重组没有影响。基于这些结果，我们得出结论，这种方法未能定义一个离散的DNA结合结构域。讨论了该结果可能的原因。
