Part:BBa_K4620005
AhyBURP
AhyBURP
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.
AhyBURP is a cyclase from the peanut (Arachis hypogaea). Its natural substrate is core peptide QPYGVYTW which is a precursor for bicyclic compound legumenin. Cyclisation occurs between tryptophan N- and glycine Cβ atoms and tyrosine O- and proline Cγ. Reaction is catalysed by copper (II) ion.
AhyBURP 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 Ahy construct with Bla (beta-lactamase) fusion tag.
Then we proceeded with solubility tests. All Bla-Ahy constructs were fully soluble.
Large scale protein expression was done for Bla-AhyBURP1. 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.
Then we proceeded to try to improve solubility for other Bla contructs via two mechanisms:
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. By adding denaturing agents like 8M urea or 6M guanidine hydrochloride to the purification buffers, we “unfold” the protein. Then, by gradually lowering the denaturing agent concentration under reducing conditions, we hope to get a correctly folded protein that can be used in enzymatic assays. As this process requires the protein be expressed in inclusion bodies, there was no need for fusion tags and we cloned our BURP proteins into pEXP NHis vectors (the His tag is for purification purposes) using Gibson assembly.
We decided to try two approaches for refolding - refolding by dilution (the denatured protein very quickly dilutes into a large volume of refolding buffer with no denaturing agent) and refolding by step-wise dialysis (the denatured protein slowly folds in a gradually less denaturing environment). AhyBURP2 formed inclusion bodies while SkrBURP2 did not. AhyBURP was refolded using both refolding strategies, however had a tendency to precipitate at higher concentrations (>3 mg/mL). We chose the dialysis protocol as the downstream purification process was simpler when dealing with smaller volumes. During the final purification with size exclusion chromatography, we observed a nice peak corresponding to the monomer protein, as opposed to aggregates.
Refolding process of NHis-AhyBURP2. Some precipitation occurred during dialysis, yet the soluble fraction was successfully purified using size exclusion chromatography, yielding a pure, monomeric protein for enzymatic reactions. Refolded AhyBURP2 protein was used to perform cyclisation reactions. Cu (II) ion is required for the cyclisation reaction to take place, therefore we added CuCl2 to the reaction mix and EDTA, a divalent metal ion chelator, to the negative control. Reactions were incubated for 24 hours at room temperature, then trypsin cleavage was performed that yielded a mix of several tryptic peptides. MALDI-TOF analysis was performed on a control sample. MS peaks correlated to the expected tryptic peptide masses, and we could identify a peak with the m/z ratio of 1626.87 which corresponds to the core peptide-containing tryptic peptide (predicted m/z = 1626.83).
We compared the core peptide-containing tryptic peptide peaks of control (pink) and reaction (blue) samples. Peak at 1624.86 can be observed for the reaction sample which is absent in the control sample. This peak is characteristic to the cyclic peptide, as 2 hydrogen atoms are lost upon the cyclisation. We can see that the produced peptide is a minor product, therefore cyclisation reaction conditions should be optimised to increase the yield of the desirable reaction product.
Next, we wanted to add separate peptides for trans cyclisation reaction. As we had problems with TEV cleavage of peptides, we added GB1-peptide conjugates to the reaction mix. We successfully identified several peaks corresponding to Ahy as well several peptide-GB1 conjugate peaks, but unfortunately tryptic peptide containing core peptide (m/z = 3344) could not be identified. Therefore we hypothesise that TEV cleavage should definitely be performed in order to achieve successful trans cyclisation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 664
Illegal SapI.rc site found at 339
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