Part:BBa_K4620011
Cca-BURP2 tryptic peptide modification (AN double insertion) containing the core peptide
NSQLLAVWR is a modification of LNSQLLVWR, a naturally occuring peptide formed after tryptic digestion of CcaBURP2 protein. Wild type protein contains core peptide fragment QLLVW. QLLVW is a peptide that can be cyclised by BURP domain protein from eastern redbud (Cercis canadensis (Part code), 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, LNSQLLVWR 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. We chose substrate a little bit longer than the core domain in order to make expression and purification easier. We designed several modificated core peptide analogues in order to see what are the limits of CcaBURP2 cyclisation capacity. In this peptide alanine and asparagine are inserted between leucine and valine. Our team cloned LNSQLLANVWR gene into pEXP-GB1 expression vector, containing GB1 (domain B1 of Immunoglobulin G-binding protein G from Streptococcus sp.), N-terminal 8xHis tag and TEV protease cleavage site.
NSQLLANVWR insert amplification using PCR
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.
GB1-NSQLLANVWR SEC on 16/600 HiLoad 75 pg and 30 pg, fractions on SDS gel
Cleavage of GB1 tag with TEV protease was carried out overnight at 4 oC. Cleaved protein was then applied to HiLoad 16/600 30 pg chromatography column. GB1 peak and two other peaks can be observed; one of them is characteristic to the cleaved peptide, other could be peptide dimer.
It can be observed that after the peptide has been cleaved off, GB1 protein migrates higher on the gel despite the loss of mass. Cleaved peptide unfortunately is too small to be observed even on 15 % tricine gel.
Gel analysis of peptide cleavage and purification
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]
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