Part:BBa_K1621006
Dihydroxyacid dehydratase (Salmonella Typhimurium)
This part contains the coding sequence of an enzyme called dihydroxyacid dehydratase (DHAD) and is derived from Salmonella Typhimurium.
DHAD is a crucial component in the biosynthesis pathway of two proteinogenic amino acids, L-isoleucine and L-valine. Natural substrates are α-, β-dihydroxy-β-methylvaleric acid (an isoleucine precursor) and α-, β-dihydroxyisovaleric acid (a valine precursor). To be able to exhibit its catalytical function the iron sulphur cluster (4Fe4S) in the core of the protein is crucial (Myers et al. 1961).
As the protein has such a crucial function in amino acid metabolism, enzymes with the same function are found in many bacterial species, including Escherichia coli. In mammalians, L-isoleucine and L-valine are so called essential amino acids, which have to be taken up by nutrition because they cannot be synthesized de novo. Thus, it is not surprising that no homologues of DHAD are described for mammalians.
Salmonella Typhimurium is a subtype of the subspecies Salmonella enterica ssp. enterica. It is responsible for more than 90% of human infections with Salmonella species and mainly affects the gastrointestinal tract. Infections are mediated by food, for example pork or decayed eggs.
In 2012, Meyer et al. described DHAD as a new specific antigen for S. Typhimurium. Regarding the problem that currently used markers for S. Typhimurium infections exhibit strong cross-reactivity in diagnostic tests, more specific immunogenic proteins are highly needed for medical applications based on antibody interactions (Nielsen et al., 1995). Hence, the same group generated a single chain variable fragment (scFv) specifically binding to DHAD.
After cloning the part into an expression vector, the protein can be efficiently overexpressed in Escherichia coli. Figure 1 shows the vector that was used for overexpression. E. coli BL21 Rosetta cells were grown in LB-medium (Luria Bertani) containing ampicillin and chloramphenicol for 3 h and the expression was induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside).
The harvested cells were lysed by sonification and proteins were separated from cell debris by ultracentrifugation. Afterwards, the target protein 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 scFv.
The protein solutions before and after affinity purification were analyzed by SDS-PAGE. Figure 2 shows that DHAD (~63 kDa) as well as the scFv (~30 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.
References
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 generation of antibodies against Salmonella Typhimurium using phage display (2012). BMC Biotechnology 12:29.
Myers JW: Dihydroxy acid dehydrase: an Enzyme Involved in the Biosynthesis of Isoleucine and Valine (1961). Journal of Biological Chemistry 236 No. 5.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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