Part:BBa_K3739000

his-hutH

Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of  changing L-histidine into trans-urocanate. Use Histidine ammonia-lyase to change L-histidine into trans-urocanate. And  his-tag is used to purify the protein.

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).

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.

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. 3. The relationship of 1/Enzyme activity and 1/concentration of L-histidine.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 168
  Illegal NgoMIV site found at 604
  Illegal NgoMIV site found at 1339
  Illegal NgoMIV site found at 1575
  Illegal AgeI site found at 241
  Illegal AgeI site found at 1068
  Illegal AgeI site found at 1564
  
• 1000
  COMPATIBLE WITH RFC[1000]