Difference between revisions of "Part:BBa K3739086"

Revision as of 17:20, 21 October 2021

his-hutH-FT

Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of changing L-histidine into trans-urocanate. The proteins labeled of tetracysteine motif tag (FlAsH tag, FT in short) can be detected with the biarsenical compound FlAsH.

Usage and Biology

Characterization

The hutH gene from Pseudomonas putida was heterologously expressed in chassis bacteria to produce histidine ammonia-lyase (HutH), which could catalyze L-histidine to trans-urocanate. Promoter (BBa_J23100), RBS (BBa_B0030), hutH gene, and terminator (BBa_B0010) were assembled into pET-28a(+) plasmid backbone to express the HutH. The constructed plasmid was transformed into Vibrio natriegens through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by regular PCR (Fig. 1) and sequencing. Then, the colony with the corrected sequence was cultivated to express HutH target protein. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the HutH from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2). The absorbance of L-histidine was measured at 277 nm (ε = 18000 (mol · L-1)-1 · cm-1) to obtain the data of concentration, which was used to calculate the kinetic constants of Km and kcat (Fig. 3). Thus, these data demonstrate that HutH works well to catalyze L-histidine to trans-urocanate. Fig. 1. The result of regular PCR. Plasmid pET-28a(+). Fig. 2. SDS-PAGE analysis of protein in lysate of Vibrio natriegens and the eluant. Target bands (55.7 kDa) can be observed at the position around 50 kDa. Fig. 3. The relationship of 1/Enzyme activity and 1/concentration of L-histidine. Sequence and Features