2. Biology Dictionary
  3. Dihydrofolate Reductase Reconstruction

Dihydrofolate Reductase Reconstruction二氢叶酸还原酶重建

释义：

The gene for DHFR is rationally dissected into two fragments called F[1,2] and F[3]. Two proteins or protein domains that are thought to bind to each other can then be fused to either of the two DHFR fragments. Reconstitution of enzyme activity can be monitored in vivo by cell survival in DHFR-negative cells grown in the absence of nucleotides. A fluorescence assay can also be carried out taking advantage of fMTX binding to reconstituted DHFR. The basis of this assay is that complementary fragments of DHFR, when expressed and reassembled in cells, will bind with high affinity (Kd 5 540 pM) to fMTX in a 1:1 complex. fMTX is retained in cells by this complex, whereas the unbound fMTX is actively and rapidly transported out of the cells. Survival depends only on the number of molecules of DHFR reassembled.

DHFR 基因被理性地切割成两个片段，称为 F[1,2] 和 F[3]。然后，认为相互结合的两个蛋白质或蛋白质域可以分别融合到这两个 DHFR 片段中的任何一个。可以通过在 DHFR 缺失的细胞中培养并在没有核苷酸的情况下观察细胞存活来监测酶活性的重建。还可以进行荧光检测，利用 fMTX 与重建的 DHFR 的结合。该检测的基础是，当 DHFR 的互补片段在细胞中表达并重新组装时，它们将以高亲和力 (Kd 5 540 pM) 与 fMTX 结合，形成 1:1 复合物。通过这个复合物，fMTX 被细胞保留，而未结合的 fMTX 会被迅速且主动地转运出细胞。生存仅取决于重组的 DHFR 分子的数量。

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