Difference between revisions of "Part:BBa K4348001"
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An enzyme that converts cholesterol to 4-cholesten-3-one. It was discovered in Eubacterium coprostanoligenes by a team at the Broad Institute of MIT in 2021. This conversion is the first of the three-step pathway of converting cholesterol to coprostanol, a sterol that does not get absorbed by the gut. A 6x his-tag is attached to the N-terminal end, allowing for easy purification and analysis through nickel/cobalt columns and western blotting. | An enzyme that converts cholesterol to 4-cholesten-3-one. It was discovered in Eubacterium coprostanoligenes by a team at the Broad Institute of MIT in 2021. This conversion is the first of the three-step pathway of converting cholesterol to coprostanol, a sterol that does not get absorbed by the gut. A 6x his-tag is attached to the N-terminal end, allowing for easy purification and analysis through nickel/cobalt columns and western blotting. | ||
| − | + | =Introduction= | |
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| + | ==Biology== | ||
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| + | ==Results | ||
| + | In order to solubilize ismA, a SUMO tag was attached on the N-terminus using Gibson assembly. The primers used were psmt_ifit_F, psmt_ifit_R, pPROEX_ismA_F and pPROEX_ismA_R. The ligations were verified via NotI and NcoI double digestion. A successful ligation was transformed into BL21, then incubated with 0.5mM IPTG at 16˚C for 24 hours to favor slower production of proteins to prevent misfolding. The proteins were extracted with B-per reagent and the soluble fraction was purified. A western blot with anti-his antibodies was performed, showing successful purification of ismA. | ||
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| + | [[File:SUMO-ismA western.png|center|500px|<strong>Anti-his Western Blot of SUMO-ismA and ismA (no SUMO) protein purification.</strong> ismA-SUMO was detected in the elution of the ismA-SUMO sample group, indicating that it was successfully purified out from the inclusion bodies. However, the only detectable ismA in the ismA (no SUMO) sample was found in the cell lysate, indicating that all the ismA was stuck completely in the inclusion bodies.]] | ||
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| + | We tested our purified, uncleaved SUMO ismA by combining it in our reaction buffer with NADP+ (ismA’s cofactor) and cholesterol, ismA’s substrate. We looked for the formation of cholestenone on the GCMS, its product. | ||
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| + | [[File:IsmA in vitro results.png|center|500px|<strong>Anti-his Western Blot of SUMO-ismA and ismA (no SUMO) protein purification.</strong> ismA-SUMO was detected in the elution of the ismA-SUMO sample group, indicating that it was successfully purified out from the inclusion bodies. However, the only detectable ismA in the ismA (no SUMO) sample was found in the cell lysate, indicating that all the ismA was stuck completely in the inclusion bodies.]] | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K4348001 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4348001 SequenceAndFeatures</partinfo> | ||
Revision as of 20:53, 13 October 2022
ismA_his
An enzyme that converts cholesterol to 4-cholesten-3-one. It was discovered in Eubacterium coprostanoligenes by a team at the Broad Institute of MIT in 2021. This conversion is the first of the three-step pathway of converting cholesterol to coprostanol, a sterol that does not get absorbed by the gut. A 6x his-tag is attached to the N-terminal end, allowing for easy purification and analysis through nickel/cobalt columns and western blotting.
Introduction
Biology
==Results In order to solubilize ismA, a SUMO tag was attached on the N-terminus using Gibson assembly. The primers used were psmt_ifit_F, psmt_ifit_R, pPROEX_ismA_F and pPROEX_ismA_R. The ligations were verified via NotI and NcoI double digestion. A successful ligation was transformed into BL21, then incubated with 0.5mM IPTG at 16˚C for 24 hours to favor slower production of proteins to prevent misfolding. The proteins were extracted with B-per reagent and the soluble fraction was purified. A western blot with anti-his antibodies was performed, showing successful purification of ismA.
We tested our purified, uncleaved SUMO ismA by combining it in our reaction buffer with NADP+ (ismA’s cofactor) and cholesterol, ismA’s substrate. We looked for the formation of cholestenone on the GCMS, its product.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 58
- 1000COMPATIBLE WITH RFC[1000]