Difference between revisions of "Part:BBa K4620012"

Revision as of 15:25, 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.

< img src="" >

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. < img src="" >

Sequence and Features