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| − | == '''anti-dihydroxyacid dehydratase (scFv)''' ==
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| − | [[File: 2015_Freiburg_pHAL14-TM228.2.3_D9.jpg|300px|thumb|right|'''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)]]
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| − | This part contains the coding sequence of a single chain variable fragment (scFv) that binds specifically to the dihydroxyacid dehydratase DHAD ([https://parts.igem.org/Part:BBa_K1621006 Bba_K1621006]) derived from ''Salmonella'' Typhimurium.
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| − | Meyer ''et al.'' (2012) showed that [https://parts.igem.org/Part:BBa_K1621006 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 to 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.
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| − | 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).
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| − | 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 imidazole. The same method was used to overexpress and purify the corresponding antigen [https://parts.igem.org/Part:BBa_K1621006 DHAD].
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| − | The protein solutions before and after affinity purification were analyzed by SDS PAGE. Figure 2 shows that the scFv (~30 kDa) as well as [https://parts.igem.org/Part:BBa_K1621006 DHAD] (~63 kDa) were efficiently enriched and successfully purified from the whole cell lysate.
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| − | Both purified proteins were used to perform Western Blot analysis to show their specific binding properties. This is visualized in figure 3.
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| − | 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.
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| − | [[File: 2015_Freiburg_SDS-PAGE_13_15.png|400px|thumb|left|'''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 30 kDa for the scFv, respectively. FT=Flowthrough, W=Wash, E=Elution, DHAD=dihydroxyacid dehydratase, scFv=single chain variable fragment. (Meyer ''et al.'', 2012)]]
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| − | [[File: 2015_Freiburg_WB_Salmonalla_gesamt.png|400px|thumb|left|'''Figure 3: Western Blot of ''S.'' Typhimurium antigen (DHAD) and ''S.'' Typhimurium antibody (anti-DHAD, [https://parts.igem.org/Part:BBa_K1621007 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 30 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.]]
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| − | ''' References '''
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| − | Meyer T, Schirrmann T, Frenzel A, Miethe S, Stratmann-Selke J, Gerlach GF, Strutzberg-Minder K, Dübel S, Hust M: Identification of immunogenic proteins and
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| − | generation of antibodies against Salmonella Typhimurium using phage display (2012). ''BMC Biotechnology'' 12:29.
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| − | Myers JW: Dihydroxy acid dehydrase: an Enzyme Involved in the Biosynthesis of Isoleucine and Valine (1961). ''Journal of Biological Chemistry'' 236 No. 5.
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| − | Nielsen B, Baggesen D, Bager F, Haugegaard J, Lind P: The serological response to Salmonella serovars typhimurium and infantis in experimentally infected pigs. The time course followed with an indirect anti-LPS ELISA and bacteriological examinations (1995). ''Vet Microbiol'' 47:205–218.
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