| Summary: | 碩士 === 國立臺北科技大學 === 生物科技研究所 === 96 === TAP tag based affinity purification has been widely used to isolation multi-subunit protein complexes. In our laboratory, calmodulin ( CaM ) conjugated with Ni-NTA ( nitrilotriacetic acid ) gold binding to CBP ( calmodulin binding peptide ) is the consistent process. Electron microscopy imaging and 3-D reconstruction of a camodulin-NTA gold labeled protein complex allows address of the interesting subunit. However, such strategy suffers from low occupany of CaM-NTA gold, paralleled with the observation that capture of TAP tagged protein complex on CaM column is also low. In order to improve these present obstacles, this dissertation designs to insert a poly-histidine tag into TAP tag. After TEV protease cutting, the interesting protein can not only do a second purification based on a NTA column, but also label NTA gold onto the 10His inserted into the C-terminus directly.
At first, pBST1479 was modified with insertion of the sequences encoding poly-histidine tag into TAP tag sequence following CBP by overlap extension based on PCR reaction. With the help of homologous recombination, the TAP ( CBP-10H ) tag was added to C-terminus of interesting protein using high efficiency yeast transformation.
In this dissertation, I select Tfg2 subunit to be the target and then attest the TAP ( CBP-10H ) tag is successfully inserted. Most importantly, the recovery of secondary purification using Ni-NTA column is much better, and Ni-NTA gold can be specifically attached to Tfg2 in the complex of RNA polymerase / TFIIF.
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