Part:BBa_K3739086
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.
Biology
HutH exists in various microorganisms in nature that could catalyze L-histidine to trans-urocanate (Fig. 1). In our project, the gene of HutH from Pseudomonas putida was expressed in chassis bacteria to produce histidine ammonia-lyase (HutH). After that, the product of trans-urocanate was further converted to glutamic acid.
HutH is found in the livers of vertebrates and bacteria, such as E. coli, Salmonella and pseudomonas, which is specific to L-histidine and can be inhibited by D-histidine or imidazole.
Usage
His-tag (6×His) is expressed at the N end of HutH for protein purification. A strong promoter (BBa_J23100) and HutH parts (RBS-HutH-Terminator) were connected to the pET-28a(+) vector by the standard assembly, which was further transformed into E. coli DH5α. After being selected by kanamycin and verified by colony PCR and sequencing, the positive colony with corrected plasmid was obtained.
Characterization
1. Agarose gel electrophoresis (AGE)
When we were building this circuit, regular PCR was used to certify that the plasmid was correct. Target bands (1877 bp) can be observed at the position around 2000 bp (’Fig. 2').
2. SDS-PAGE
The plasmid verified by sequencing was successfully transformed into '’Vibrio natriegens. After being cultivated and induced by ITPG, GE AKTA Prime Plus FPLC System was employed to get purified protein from broken supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in SDS-PAGE of HutH-his (Fig. 3), the target protein (55.7 kDa) could be observed at the position around 50 kDa on the purified protein lanes (FR), but not in the negative control groups (NC).
3. Enzyme kinetic constants measurement
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. 4 and Table 1)
Fig. 1. Schematic diagram of HutH catalytic process.
Fig. 2. The result of regular PCR. Plasmid pET-28a (+).
Fig. 3. 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. 4. The relationship of 1/Enzyme activity and 1/concentration of L-histidine.
Table 1. Enzyme kinetic constants with L-histidine
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 162
Illegal NgoMIV site found at 598
Illegal NgoMIV site found at 1333
Illegal NgoMIV site found at 1569
Illegal AgeI site found at 235
Illegal AgeI site found at 1062
Illegal AgeI site found at 1558 - 1000COMPATIBLE WITH RFC[1000]
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