| 摘要 |
A method to aid in the efficient construction of fusion proteins involving multiple domains is provided in which a new expression vector is created to allow rap id generation of a library of cassettes. Cassettes have a standard vector structure based on fo ur specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesi ve end restriction enzymes, they can be fused in any order and number of times. Furthermore, th e insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of a detectable activity . The utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.
|
