Difference between revisions of "Part:BBa K3739086"
| Line 10: | Line 10: | ||
===Characterization=== | ===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 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.<br/> |
| − | 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 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'').<br/> |
| − | 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). | + | 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). <br/> |
| − | Thus, these data demonstrate that HutH works well to catalyze L-histidine to trans-urocanate. | + | Thus, these data demonstrate that HutH works well to catalyze L-histidine to trans-urocanate.<br/> |
| − | ''Fig. 1''. The result of regular PCR. Plasmid pET-28a(+). | + | ''Fig. 1''. The result of regular PCR. Plasmid pET-28a(+).<br/> |
| − | ''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. 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.<br/> |
''Fig. 3''. The relationship of 1/Enzyme activity and 1/concentration of L-histidine. | ''Fig. 3''. The relationship of 1/Enzyme activity and 1/concentration of L-histidine. | ||
<!-- --> | <!-- --> | ||
Revision as of 17:22, 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
- 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]