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Team:TU-Delft/SUMOCleavageUlp
From 2013.igem.org
Peptide production
Figure 1: Schematic diagram of the peptide production
The era of antibiotics are now outdated as the microorganisms started gaining new resistance mechanism to evade the effects of antibiotics. Nowadays more and more researches are focused towards new antimicrobial that can be more effective against these pathogens.
The antimicrobial peptides (AMP's), natural or de-novo are becoming more popular due to their potential to kill or restrict the growth of microorganisms. They form a part of chemical defense against predators. All organisms, including higher order eukaryotes have some antimicrobial peptides that acts against specific strains of microorganisms.They are usually charged and interact with the opposite charges present on the membrane thus boring a hole through the membrane subsequently lysing the cell.Unlike, antibiotics they do not interfere in the genetic, transcriptional or translational machinery of the cells.
We chose 3 AMP's that are mostly produced in the skins of toads and frogs. They are magainin from Xenopus laevis , signiferin from Crinia signifera and maximin-H5 from Bombina maxima. The reasons for choosing these AMP's could be attributed to their target specific nature, charge and length of peptides. They do not affect humans, which makes them a suitable candidate as pharmaceuticals.
The recombinant production of these peptides in E. coli would help large scale production of these peptides and administer them as antimicrobials. But, the major hurdle in producing them in vivo is the formation of inclusion bodies as they are charged. This could be overcome by making protein fusion with SUMO (Small Ubiquitin like Modifier), MBP (Maltose Binding Protein), Trx (Thioredoxin) etc.
In this project, a SUMO-peptide fusion was opted as a suitable expression system where the enzyme Ulp cleaving the fusion is also expressed and controlled by a negative transcriptional cascade. This fusion increases the solubility of the peptide and reduces inclusion bodies formation(Figure 1).
The in-vivo production of peptides at sufficient quantities is crucial to kill the MRSA. To prove that we produce enough peptides we carried out the following characterization experiments.
SUMO cleavage by Ulp1
Cleavage of SUMO from Peptide by Ulp-1 protease.
The SUMO-peptide helps in increasing the soluble fraction of peptide but peptides are not biologically active in a fusion, they have to be cleaved from the fusion to get a active peptide fraction. This was achieved by in vivo production on SUMO specific Ulp-1 proteases.
A gene was constructed in such a way that the SUMO-peptide production was driven by the strong T7 phage promoter and the Ulp-1 production was driven by arabinose inducible promoter pBad. The plasmid was transformed into an BL21(DE3) pLysS strain. This construct was designed to check whether in vivo cleavage is possible. The main idea of the experiment is to first produce large amount of soluble fraction of SUMO-peptides and the produce the Ulp protease to cleave the sufficiently produced fusion peptides.
Figure 1: Part BBa_K1022104/5/6
