Difference between revisions of "Part:BBa K3739000"
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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.<br/> | 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.<br/> | ||
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.<br/> | 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.<br/> | ||
| − | [[File:T--XMU-China--his-Uro.png|200px|alt text]] | + | <center>[[File:T--XMU-China--his-Uro.png|200px|alt text]]</center><br/> |
'''Fig. 1'''. Schematic diagram of HutH catalytic process.<br/> | '''Fig. 1'''. Schematic diagram of HutH catalytic process.<br/> | ||
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====1. Agarose gel electrophoresis (AGE)==== | ====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''').<br/> | 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''').<br/> | ||
| − | [[File:T--XMU-China--K3739031_.png|200px|alt text]] | + | <center>[[File:T--XMU-China--K3739031_.png|200px|alt text]]</center><br/> |
'''Fig. 2'''. The result of regular PCR. Plasmid pET-28a (+).<br/> | '''Fig. 2'''. The result of regular PCR. Plasmid pET-28a (+).<br/> | ||
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====2. SDS-PAGE==== | ====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).<br/> | 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).<br/> | ||
| − | [[File:T--XMU-China--K3739031_SDS.png|200px|alt text]] | + | <center>[[File:T--XMU-China--K3739031_SDS.png|200px|alt text]]</center><br/> |
'''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.<br/> | '''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.<br/> | ||
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====3. Enzyme kinetic constants measurement==== | ====3. Enzyme kinetic constants measurement==== | ||
The absorbance of ''L''-histidine was measured at 277 nm (ε = 18000 (mol · L<sup>-1</sup>)<sup>-1</sup> · cm<sup>-1</sup>) to obtain the data of concentration, which was used to calculate the kinetic constants of Km and ''kcat'' ('''Fig. 4''' and '''Table 1''')<br/> | The absorbance of ''L''-histidine was measured at 277 nm (ε = 18000 (mol · L<sup>-1</sup>)<sup>-1</sup> · cm<sup>-1</sup>) to obtain the data of concentration, which was used to calculate the kinetic constants of Km and ''kcat'' ('''Fig. 4''' and '''Table 1''')<br/> | ||
| − | [[File:T--XMU-China--HutH_Enzyme_Activity.png|200px|alt text]] | + | <center>[[File:T--XMU-China--HutH_Enzyme_Activity.png|200px|alt text]]</center> |
'''Fig. 4'''. The relationship of 1/Enzyme activity and 1/concentration of ''L''-histidine.<br/> | '''Fig. 4'''. The relationship of 1/Enzyme activity and 1/concentration of ''L''-histidine.<br/> | ||
| − | [[File:T--XMU-China--000table1.png|360px]]<br/> | + | <center>[[File:T--XMU-China--000table1.png|360px]]</center><br/> |
'''Table 1'''.Enzyme Kinetic constants with ''L''-histidine<br/> | '''Table 1'''.Enzyme Kinetic constants with ''L''-histidine<br/> | ||
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Latest revision as of 03:52, 22 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.
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.
Fig. 1. Schematic diagram of HutH catalytic process.
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).
Fig. 2. The result of regular PCR. Plasmid pET-28a (+).
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).
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.
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. 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 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]