BBa_K1621007 1 BBa_K1621007 anti-dihydroxyacid dehydratase 2015-08-28T11:00:00Z 2015-10-26T10:24:41Z 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). <br> <br> This part contains the coding sequence of a single chain variable fragment (scFv) that binds specifically to dihydroxyacid dehydratase (DHAD) of ''Salmonella'' Typhimurium. <br> <br> 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. <br> <br> 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. <br> 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. <br> 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. <br> <br> Both purified proteins were used to perform Western Blot analysis to show the specific binding properties. This is visualized in figure 3. <br> <br> 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. <br> false false _2038_ 25634 25598 9 false 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. false annotation2440557 1 linker region range2440557 1 385 438 annotation2440558 1 VL anti-dihydroxyacid dehydratase range2440558 1 439 807 annotation2440547 1 VH anti-dihydroxyacid dehydratase range2440547 1 4 384 igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z