Difference between revisions of "Part:BBa K3739000"
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===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/> |
| + | '''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/> |
| − | 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. 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 17:28, 21 October 2021
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.
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).
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.
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
- 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 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 - 1000COMPATIBLE WITH RFC[1000]