Difference between revisions of "Part:BBa K4726001"
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===Characterization and results=== | ===Characterization and results=== | ||
| − | The protein was expressed using 0.1 mM IPTG induction in LB broth. After 18 hours of induction at 26 °C, protein were extracted and purified. In well #6, we see overproduction of the aforementioned protein (119 | + | The protein was expressed using 0.1 mM IPTG induction in LB broth. After 18 hours of induction at 26 °C, protein were extracted and purified. In well #6, we see overproduction of the aforementioned protein (119.530 KDa). |
https://static.igem.wiki/teams/4726/wiki/results/sds-page-purif-wash-elution-igem.png | https://static.igem.wiki/teams/4726/wiki/results/sds-page-purif-wash-elution-igem.png | ||
| − | Figure 1.SDS-PAGE electrophoresis of purification steps. | + | |
| + | <b>Figure 1</b>.SDS-PAGE electrophoresis of purification steps. | ||
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Latest revision as of 01:20, 10 October 2023
α-D-galactosamine galactosaminidase_Flavonifractor plautii
Usage and Biology
This encodes a α-D-galactosamine galactosaminidase from Flavonifractor plautii originating from the human gut microbiota. This enzyme is available on Uniprot under the entry P0DTR5. We added a 6X his-tag at the C-terminus end of the protein.
This enzyme works in pair with P0DTR4 (BBa_K4726000) to fully remove the A antigen. It removes the residual α-D-galactosamine that is left after P0DTR4 takes action. P0DTR5 is not active towards A antigen without prior reaction with P0DTR4. The use of both P0DTR4 and P0DTR5 leads to the production of the H antigen, most commonly known as O type blood.
The expression of this enzyme led to significant proteotoxicity shown by an impaired growth of Escherichia coli BL21 (DE3). We thus recommended to use Escherichia coli Rosetta (DE3) pLysS which helps stabilizing pET recombinants encoding proteins.
Characterization and results
The protein was expressed using 0.1 mM IPTG induction in LB broth. After 18 hours of induction at 26 °C, protein were extracted and purified. In well #6, we see overproduction of the aforementioned protein (119.530 KDa).
Figure 1.SDS-PAGE electrophoresis of purification steps.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 865
Illegal EcoRI site found at 1411 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 865
Illegal EcoRI site found at 1411 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 865
Illegal EcoRI site found at 1411 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 865
Illegal EcoRI site found at 1411 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 865
Illegal EcoRI site found at 1411
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1894 - 1000COMPATIBLE WITH RFC[1000]
References
[1] Rahfeld P, Sim L, Moon H, Constantinescu I, Morgan-Lang C, Hallam SJ, Kizhakkedathu JN, Withers SG. An enzymatic pathway in the human gut microbiome that converts A to universal O type blood. Nat Microbiol. 2019 Sep;4(9):1475-1485. doi: 10.1038/s41564-019-0469-7. Epub 2019 Jun 10. PMID: 31182795.