Difference between revisions of "Part:BBa K1621007"

Revision as of 12:11, 31 August 2015

anti-dihydroxyacid dehydratase (scFv)

Figure 1: Expression vector for scFv specifically binding DHAD. scFv was expressed with C-terminally fused His- and c-Myc-tag. scFv=single chain variable fragment, DHAD=dihydroxyacid dehydratase.(Meyer et al., 2012)

This part contains the coding sequence of a single chain variable fragment (scFv) that binds specifically to dihydroxyacid dehydratase DHAD (Bba_K1621006) derived from Salmonella Typhimurium.

Meyer et al. (2012) showed that DHAD acts as a specific antigen for S. Typhimurium. This subtype of the Salmonella enterica subspecies enterica causes more than 90% of all Salmonella infections. scFvs binding specifically against DHAD were identified by phage display using the human naïve antibody library HAL7/8. For the scFv that is encoded by this part (TM228.2.3-D9) an EC[50] value of 50 nM was determined by titration ELISA and the binding to DHAD was verified by immunoblotting.

After cloning the part into an expression vector, the scFv can be efficiently overexpressed in Escherichia coli. Figure 1 shows the vector that was used for overexpression. E. coli C43 cells were cultured in LB medium (Luria Bertani) containing ampicillin and chloramphenicol over night at 30°C and the expression was induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside). The harvested cells were lyzed by sonification and proteins were separated from cell debris by ultracentrifugation. Afterwards, the scFv was purified by affinity chromatography. The His-tag that is C-terminally fused to the protein specifically binds to Ni-NTA agarose beads and is eluted with 500 mM imidazol. The same method was used to overexpress and purify the corresponding antigen DHAD. The protein solutions before and after affinity purification were analyzed by SDS PAGE. Figure 2 shows that the scFv (~30 kDa) as well as DHAD (~63 kDa) were efficiently enriched and successfully purified from the whole cell lysate.

Both purified proteins were used to perform Western Blot analysis to show their specific binding properties. This is visualized in figure 3.

The part was shipped to the registry in standard pSB1C3, beginning with a start codon (ATG). It was inserted into the shipping backbone by Gibson Assembly and the sequence was verified afterwards.

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 500 mM Imidazole. The expected molecular weights for the proteins are 63 kDa for DHAD and 37 kDa for the scFv, respectively. FT=Flowthrough, W=Wash, E=Elution, DHAD=dihydroxyacid dehydratase, scFv=single chain variable fragment. (Meyer et al., 2012)

Figure 3: Western Blot of S. Typhimurium antigen (DHAD) and S. Typhimurium antibody (anti-DHAD, scFv). (A) Western Blot of His-tagged DHAD as well as the corresponding scFv with anti-His HRP Conjugate. The expected molecular weights for the proteins are 63 kDa for DHAD and 37 kDa for the scFv, respectively. (B) Western Blot of DHAD with the purified S. Typhimurium scFv was used in a 1:100 dilution. Additionally, the scFv is c-Myc tagged. Anti-c-Myc antibody (1:1000; rabbit) was used in a second step. For detection, the anti-rabbit HRP antibody (1:5000) was used.