Part:BBa_K1394000

SUMO fusion domain with Magainin 1 star peptide

This contruct codes for a protein generating device with an N-terminal SUMO fusion domain preceded by a 6x-HIS tag. The design of the expression vector is based on the SUMO peptide sequence reported by TU Delft 2013 iGEM. Specifically, the expression system is an improvement and modification of the TU Delft 2013 protein expression system cataloged in the parts registry at BBa_K1022101

We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:

1. Standardisation of the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrickBBa_K525998. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.
   
2. Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,BBa_K731721).
3. The original biobrick BBa_K1022101 codes for three amino acid residues before the HIS-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore were not included.
   
4. The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.

Note that any protein coding region can be cloned into this BioBrick expression vector by taking advantage of the AgeI site in SUMO present at base pairs 355-360 (ACCGGT). The cloning strategy would be to use the AgeI site at the 5' end of the sequence and any restriction site in the BioBrick suffix at the 3' end (SpeI or PstI). Note when cutting the SUMO sequence with AgeI, the last codon of the SUMO protein (GGT) is removed. Therefore any insert which is being cloned into the vector needs to have a 5' GGT immediately after the AgeI restriction site. Also, cutting at the BioBrick suffix will remove the BioBrick terminator currently in the gene. Thus, the insert must also contain a compatible terminator sequence.

Protein coding region

The protein coding region codes for a peptide with the following sequence: MHHHHHHSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMDDNDIIEAHREQTGG GIGKFLHSAGKFGKAFVGEIMKS C GIGKF LHSAGKFGKAFVGEIMKS IDGR GIGKFLHSAGKFGKAFVGEIMKS C GIGKFLHSAGKFGKAFVG EIMKS

The SUMO peptide ends at residues EQTGG.

The protein following SUMO is a star peptide, using antimicrobial peptides(AMPs) as arms. AMPs are small, approximately 50 residue peptides secreted by some bacterial and eukaryotic cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells. AMPs have been recombinantly expressed in a number of organisms, including E. coli and B. Subtilis.

Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (N. Wiradharma, S. Q. Liu and Y. Y. Yang, Small, 2012, 8, 362-366) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighbouring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (for AMP sequence, see M. Zasloff, Proc. Natl. Acad. Sci. U. S. A., 1987, 84, 5449-5453). Magainin 1 peptides will be placed to the ends of each star arm.

Magainin 1 was chosen because the Magainins are one of the major classes of antimicrobial peptides, being well studied and characterised. In addition, we were concerned that tethering the antimicrobial peptide to the star peptide might interfere with its antimicrobial activity. Magainin 1, however, has previously been tethered to surfaces, where it has imparted the surfaces with microbicidal properties (Glinel et al., 2008; Humblot et al., 2009). We surmised that if Magainin 1 maintained its activity while anchored to surfaces, it may also maintain its activity while anchored to a star peptide.

The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.

When attached to a star peptide it will have the following structure:

There are several design elements to note:

• The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.
• The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.

See the University of Melbourne 2014 iGEM team project page for further background on the design rationale of the peptide.

DNA characterization

The part was a product of PCR cloning into pSB1C3 (see team page for thorough discussion of the cloning approach).

After a digestion with EcoRI and PstI, the empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps; see lane C2* for this BioBrick).

N.B. MW marker is the 100 bp Ladder from Axygen.

The DNA sequence listed on the registry was confirmed using Sanger sequencing at the Australian Genome Research Facility. 

Protein characterization

The plasmid was transformed to both SHuffle T7 and BL21(DE3) cells, and a single colony was cultured and induced overnight at 17 °C. A whole cell sample both before IPTG induction (-IPTG) and after the induction period (+IPTG) were boiled in SDS-PAGE sample buffer and loaded on a 15% tris-glycine gel. The whole cell Coomassie stained gel is shown below alongside the NEB P7712S molecular weight marker (see lanes labelled Mag1 Star for this BioBrick).

A small-scale purification was carried out using the HIS-tag at the N-terminus 

After purification, a concurrent Western Blot and Coomassie stain on both the pre-induction samples from above and the purified protein (namely, the first elution from the batch purification) were run.

The Western Blot used mouse monoclonal antibodies against the N-terminal HIS-tag as primary antibodies and anti-mouse secondary antibodies (again see lanes labelled Mag1 Star):

The corresponding Coomassie stain is show below (see lanes labelled Mag1 Star:)

Note that the protein runs near its predicted molecular weight of 22.15 kDa

The sequence was probed using an in gel digestion of the bands corresponding to the protein, followed by LC-MS/MS:.

The mass spec coverage for the Magainin 1 Star Peptide expressed in SHuffle cells was as follows:

The coverage for the same peptide expressed in BL21(DE3) was found to be:

This analysis suggests that the BioBrick leads to protein expression, including elements of the SUMO fusion protein.