Team:Freiburg/Project/pRIG15 13
pRIG15_13
To generate a single chain variable fragment against dihydroxyacid dehydratase the group of Prof. Dr. Hust used a human naive antibody gene library. 1) They thankfully provided us with the sequence of the anti-dihydroxyacid dehydratase already cloned into an expression vector.
To insert the sequence for the Salmonella antibody (anti-dehydroxyacid dehydratase) into pSB1C3, we designed Gibson primers with compatible overhangs that also included the start codon ATG. This fragment was amplified via PCR from the expression vector we obtained from Prof. Dr. Hust and then assembled with the digested pSB1C3 backbone using Gibson Assembly. To prove correct insertion of our fragment we performed a test digest and verified the part by sequencing.
We used the plasmids sent to us by Prof. Dr. Hust to express both proteins, Salmonella dihydroxyacid dehydratase and anti-dihydroxyacid dehydratase, in E.coli. We could show overexpression by SDS-PAGE (figure 2) verified the interaction of both proteins by Western Blot (figure 3).
Figure 2: 12,5% SDS-PAGE analysis of the protein purification of S. Typhimurium antigen (dehydroxyacid dehydratase) and S. Typhimurium antibody (anti-dehydroxyacid dehydratase). Protein purification was performed with gravity flow columns and Ni-NTA Agarose. The protein was eluated by 500mM Imidazole. The expected molecular weight for the S. Typhimurium antigen is 63 kDa and 37 kDa for the antibody, respectively. FT-Flowthrough, W-Wash, E-Elution.
Figure 3: Western Blot of S. Typhimurium antigen (dehydroxyacid dehydratase) and S. Typhimurium antibody (anti-dehydroxyacid dehydratase). (A) Western Blot of the antigen as well as of the antibody with anti-His HRP Conjugate. Both protein are His-tagged. The expected molecular weight for the S. Typhimurium antigen is 63 kDa and 37 kDa for the antibody, respectively. (B) In this Western Blot the S. Typhimurium antigen was analyzed by 12,5% SDS-PAGE. Purified S. Typhimurium antibody was used in a 1:100 dilution. Additionally, this single chain antibody is fused to a c-Myc Tag. We used an anti-c-Myc antibody (1:1000; rabbit). For detection the anti-rabbit HRP antibody (1:5000) was used.
Link to Registry: BBa_K1621007
