Difference between revisions of "Part:BBa K4620012"
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Obtained plasmid was transformed into T7 Express competent E. coli cells and plated on LB agar plate containing ampicillin. One colony was inoculated, at OD600 = 0.8 protein expression was induced using 0.2 mM IPTG. Expression was carried out overnight at 18 oC. | Obtained plasmid was transformed into T7 Express competent E. coli cells and plated on LB agar plate containing ampicillin. One colony was inoculated, at OD600 = 0.8 protein expression was induced using 0.2 mM IPTG. Expression was carried out overnight at 18 oC. | ||
Protein was purified with Ni-NTA chromatography in 8 M urea-containing buffer and refolded after chromatography. Refolding was carried out overnight at 4oC. Folded protein was applied to HiLoad 16/600 75 pg chromatography column. | Protein was purified with Ni-NTA chromatography in 8 M urea-containing buffer and refolded after chromatography. Refolding was carried out overnight at 4oC. Folded protein was applied to HiLoad 16/600 75 pg chromatography column. | ||
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Revision as of 15:26, 12 October 2023
CcaBURP2 core peptide
QLLVW is a peptide that can be cyclised by BURP domain protein from eastern redbud (Cercis canadensis (BBa_K4620007), forming stephanotic acid. This side chain macrocyclisation reaction is catalysed by copper (II) ions. Cyclisation happens between the Cβ of leucine and indole cycle 6- position of tryptophan residue. As of this day, mechanism of this reaction has not been fully characterised.
In the nature, QLLVW is conjugated to the N-terminal domain of Cca-BURP. iGEM 2023 team Latvia-Riga produced this peptide separately in order to make trans-cyclisation reaction where the cyclisation substrate is not attached to the catalytic domain.
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Obtained plasmid was transformed into T7 Express competent E. coli cells and plated on LB agar plate containing ampicillin. One colony was inoculated, at OD600 = 0.8 protein expression was induced using 0.2 mM IPTG. Expression was carried out overnight at 18 oC.
Protein was purified with Ni-NTA chromatography in 8 M urea-containing buffer and refolded after chromatography. Refolding was carried out overnight at 4oC. Folded protein was applied to HiLoad 16/600 75 pg chromatography column.
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Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]