Part:BBa_K1394001

SUMO fusion domain with linear Magainin 1

This contruct codes for a protein generating device with an N-terminal SUMO fusion domain preceded by a 6x-HIS tag. 

The protein following this fusion tag is a sequence which codes for the antimicrobial peptide Magainin 1. 

This gene was synthesized by GenScript, as a gene for Magainin 1 was not previously available in the Registry of Standard Parts.

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 characterization

The protein coding region sequence is: MHHHHHHSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMDDNDIIEAHREQTGG GIGKFLHSAGKFGKAFVGEIMKS, with Magainin 1 sequence beginning at GIGKFLH

The plasmid was transformed to 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 rightmost lanes labelled Linear Mag1 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 the rightmost lanes labeled Linear Mag1).

The corresponding Coomassie stain is show below (see rightmost lanes:)

The band in the post-IPTG/purification lanes around 17 kDa corresponds to the linear Magainin 1 construct, and is consistent with its predicted molecular weight of 14.3 kDa.

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

We found that the Linear Magainin 1 peptide appeared to be present in the approximately 17 kDa post-purification band, with the following detected tryptic fragments in the sequence below (bold; basic residues underlined):

This suggests protein was expressed using this BioBrick device.