Difference between revisions of "Part:BBa K1621006"

Revision as of 12:04, 31 August 2015

Dihydroxyacid dehydratase (Salmonella Typhimurium)

Figure 1: Expression vector for DHAD. DHAD was overexpressed using the common expression vector pET21a+. DHAD=dihydroxyacid dehydratase, MCS=multiple cloning site (Meyer et al., 2012).

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 exhibits 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 swine meet or decayed eggs.

In 2013, 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 (Bba_K1621007).

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 lyzed 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 imidazol. 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.

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 weight of DHAD is 63 kDa 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.

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 weight 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)

Sequence and Features