Difference between revisions of "Part:BBa K4620010"

Latest revision as of 15:48, 12 October 2023

CcaBURP2 tryptic peptide containing the core peptide

LNSQLLVWR is a peptide formed after tryptic digestion of CcaBURP2 protein. It 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. Our team cloned LNSQLLVWR 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.

LNSQLLVWR insert amplification using PCR

Obtained plasmid was transformed into T7 Express competent E. coli cells and plated on LB agar plate containing ampicillin. Expression tests were carried out to determine best expression conditions.

Samples with highest expression yields were then subjected to solubility testing using BugBuster kit. Cells were lysed, samples (T = total) were taken. Lysate was centrifugated and supernatant (S = soluble) samples were taken. It can be seen that despite the fusion tag, most of the expressed protein is insoluble.

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-LNSQLLVWR SEC on 16/600 HiLoad 75 pg column

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.

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