This part is the coding sequence (CDS) of a cytoplasmic protein TupA (GenBank: BAB07375.1) with hisdine-tag at the C terminal, which catalyzes the conversion of glucuronic acid and L-glutamic acid to polyglucuronic acid and poly-γ-L-glutamic acid.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 969
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI site found at 789
Illegal BsaI.rc site found at 1181
This part C125-TupA-6His is developed from part C125-TupA,which add a his-tag at the C-terminus of TupA so that we can gain this cytoplasmic protein by histidine-labeled affinity column chromatography, and analysis at the protein level is easier.
We transfered C125-TupA-6His and C125-TupA into Bacillus subtilis SCK6 respectively, then screened by kanamycin. Transformants were grown in LB broth. Then obtained total protein by ultrasonication. performed histidine-tagged affinity column chromatography to isolate fusion protein, then verified by SDS-PAGE and Coomassie blue staining. (Fig.1)
Fig.1. SDS-PAGE analysis of total protein and the product of protein purification.M: marker ladder; Lane 1: total protein of C125-TupA-6His transformant; Lane2: the product of protein purification of C125-TupA-6His transformant; Lane3: total protein of C125-TupA transformant; Lane4: the product of protein purification of C125-TupA transformant. It can be seen that the histidine-tagged fusion protein was successfully expressed. Lane 2 showed the band corresponded with the molecular weight of TupA(57.3kDa), while lane 4 had no band.