A series of bacterial co-expression vectors with rare-cutter recognition sequences.

摘要: The bacterial co-expression method is a useful protein expression technique to reconstitute hetero-oligomeric complex in vitro. However, during the plasmid subcloning for co-expression, unintended cleavage at sequences of target cDNAs becomes more frequent as number DNA inserts increases. This problem also makes it difficult change combination targeted proteins after preparing certain construct. To avoid this problem, we have developed series vectors, which each translation cassette can be subcloned set rare-cutter restriction enzyme sites. We selected 9 different enzymes that recognize 7 or 8-base-pair sequence, and constructed 27 kinds cloning vectors 3 utilizing recognition multi-cloning Using vector system, co-expressed co-purified 7-subunit composed mammalian 26S proteasome regulatory subunits RPT1 RPT6, their associated factor, gankyrin. verified presence all by western blotting, taking advantage system fused with broad repertoire epitope tags.