Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Date created: 2015-08-28 11:00:00
Date modified: 2015-10-26 10:24:41
anti-dihydroxyacid dehydratase
| Types | DnaRegion |
| Roles | Other sequence_feature |
Description
This part contains the coding sequence of a single chain variable fragment (scFv) that binds specifically to dihydroxyacid dehydratase (DHAD) of 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. Specifically binding scFvs against DHAD were identified by phage display using the human na??ve 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 and the conditions for growth of the bacteria and induction of the expression are summarized in table 1.
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 has been 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 the specific binding properties. This is visualized in figure 3.
The part was shipped to the registry in standard pSB1C3, starting with a start codon (ATG). It was inserted into the shipping backbone by Gibson Assembly and the sequence was verified afterwards.
Notes
At position 15 a base change from guanine to thymine was inserted by site-directed mutagenesis to remove the recognition site for PstI without causing a change in the amino acid sequence.Source
The part's sequence as well as an expression plasmid carrying a His-tagged variant was obtained from the group of Prof. Dr. Hust (TU Braunschweig).| Sequence Annotation | Location | Component / Role(s) |
| VH anti-dihydroxyacid dehydratase linker region VL anti-dihydroxyacid dehydratase | 4,384 385,438 439,807 | feature/cds CDS CDS feature/cds CDS feature/cds |