Part:BBa_K4620007

CcaBURP2

BURP domains are a class of plant proteins that catalyse the cyclisation of small peptidic motifs called core sequences. These recently described cyclases have the potential to be used as biosynthetic catalysts for the preparation of cyclic peptide therapeutics, antibacterials and insecticides. Our goal is to produce functional recombinant BURP domain proteins from various plant species in order to perform cyclisation of various peptide substrates, both encoded as core peptides N-terminal of the BURP domain (cis cyclisation) and linear peptides added separately to the BURP domain protein (trans cyclisation). This could become a useful platform to produce cyclic peptides for various application purposes.

CcaBURP2 is a cyclase from eastern redbud (Cercis canadensis). Its natural substrate is core peptide QLLVW which is a precursor for stephanotic acid. Cyclisation reaction is catalysed by copper (II) ion, and in this reaction a covalent bond is formed between the Cβ of leucine and indole cycle 6- position of tryptophan residue.

CcaBURP2 was cloned into several pEXP-expression vectors containing varous fusion tags. We proceeded with small-scale expression tests in E. coli T7 express cells, combining the BURP proteins with various fusion tags, such as MBP (maltose-binding protein), Bla (beta lactamase), GST (glutathione S transferase), IF2 (initiation factor 2), GB1, LIPO-tag. The expression was induced with 0.1 mM IPTG and was left overnight at 20 ⁰C. Best expression was observed for CcaBURP2 construct with MBP and Bla fusion tags.

Large scale protein expression was done for Bla-SkrBURP, Bla-AhyBURP1, Bla-CcaBURP1, MBP-CcaBURP2. Briefly, the purification steps included cell lysis by sonication, NiNTA chromatography, TEV cleaving of the fusion tag and SEC (size exclusion chromatography). Interestingly, although the Bla fusion tag imparted great solubility, it had a tendency to be rapidly proteolytically degraded during lysis and NiNTA chromatography, which could be seen on SDS-PAGE. Unfortunately, the remaining BURP protein most likely aggregated and/or precipitated and by the last purification step of gel filtration only the Bla fusion tag was left.

Fortunately, we did not encounter the same cleaving issue with the MBP tag, therefore we concluded this was the best fusion tag for BURP domain protein production. The MBP tag could be successfully cleaved off using TEV protease. We did, however, identify a significant problem at the last purification step, while the MBP tag could be separated as a nice peak in the SEC, the CcaBURP2 protein eluted throughout the whole elution volume of the SEC column in a trailing peak, indicating an incorrectly misfolded conformation and aggregation. This is most likely due to incorrect disulfide bond formation during bacterial expression, as these bonds are important for the structural integrity of the catalytic domain.

Figure 4. SDS-PAGE gel analysis of MBP-CcaBURP2 and Bla-SkrBURP2 TEV cleaving (T - total, S - soluble) and subsequent SEC chromatograms. Most of the SkrBURP2 has precipitated and while CcaBURP2 remains in the soluble fraction, it does not elute properly in SEC.

During bacterial expression, BURP protein disulfide bonds are not properly formed which leads to incorrect protein folding. Such a protein is not active and cannot be used for enzymatic assays. Therefore, we decided to focus on two strategies to get an active protein: 1)We tried expressing the proteins in E. coli strains which are more suited for disulfide bond formation - Origami2(DE3) and SHuffle T7 express strains. 2)We tried expressing the protein in inclusion bodies and refolding.

1) Origami and Shuffle expression. These strains express certain disulfide isomerases which help the correct formation of disulfide bonds during protein expression. We hoped that by expressing in these strains we could get correctly folded BURP domain proteins. We noticed much slower growth of these cell strains. After inducing protein expression with 0.1 mM IPTG and letting cells incubate overnight, the final biomass yield was very low. The protein yield was also very low, compared to expression in T7 express cells.

To increase the protein yield, we decided to try another method for inducing protein expression - autoinduction. This method requires that both glucose and lactose be present in the cell growth medium. Once the cells have used up all the glucose, they start metabolizing lactose, at which point expression is induced. This might work better because the resulting expression induction is much slower and more gradual than IPTG induction, which puts less strain on the cells. Using this method we got a lot more biomass at the end of expression. The protein yield was also much greater per liter of medium.

Comparison of NiNTA chromatograms for MBP-CcaBURP2 expressed in SHuffle cells using IPTG induction and autoinduction.

2)We also tried refolding MBP-CcaBURP2 and obtained pure CcaBURP2 after TEV cleaving.

Purification of refolded MBP-CcaBURP2, TEV cleaving and reverse NiNTA, obtaining pure CcaBURP2. In order to further enhance correctly folded BURP domain protein production, we wanted to try cell-free synthesis as it is a fast and tunable protein production method. We cloned the CcaBURP2 gene into pETMCSI vector which was transformed into E. coli T7 express cells. Several plasmids from different colonies were isolated and control digestion reaction was performed to confirm that ligation reaction was successful.

After correct plasmids were obtained, we set up a cell-free synthesis reaction. CcaBURP2-containing pETMCSI plasmid was put into a reaction mix containing S30 E. coli lysate, amino acids, nucleotides, cofactors and energy substrates. Reaction was carried out at 30 ⁰C for 18 hours, during which precipitation occurred in the reaction mix. When we separated soluble and insoluble fractions, we saw that large amount of CcaBURP2 gene was produced, but almost all of the protein was insoluble. Therefore we solubilized the protein in a buffer containing 8 M urea, applied it to Ni-NTA column and collected an eluted fraction which was then refolded using stepwise dialysis protocol. Afterwards protein was purified using SEC chromatography.

We saw that we can successfully produce Cca2BURP using cell-free synthesis, therefore we set up more reactions to explore different reducing conditions to possibly make more soluble protein or larger amounts of it. In the previous reaction we used 1.7 mM DTT. This time we added either beta-mercaptoethanol, mix of oxidized and reduced glutathione, or glutathione mix with the addition of DTT. We saw that DTT/gluathione mix gave by far the highest yield, and higher oxidized/reduced glutathione ratio gave best results. Unfortunately none of these additives seem to enhance the solubility of the protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 154