Proteins

We have chosen two different ways to target the parasite and prevent the malaria transmission through mosquitos. Our engineered bacteria could express either an immunotoxin, or two p-proteins, or even both for maximum efficiency.

The Immunotoxin is one of our tools to block transmission of malaria parasite in mosquitos. It is composed of two main parts : The first one is a single-chain antibody fragment (scFv) directed to Pbs2l, which is a surface membrane protein of Plasmodium berghei . The second part is a lytic peptide, Shiva-1, which acts by forming “pores” on the parasite’s membrane. The immunotoxin is supposed to specifically target and lyse the parasite.

The different domaines of the immunotoxin. For more details and the sequence see the background section.

We tested expression of the immunotoxin in E.Coli (see Materials and Methods for details). In a western blot analysis of whole cell lysates we could see bands corresponding to full length immunotoxin and possibly degraded fragments of the protein (see figure). The immunotoxin contains a PelB sequence that targets it for secretion into the periplasm. We concentrated the supernatant of both the immunotoxin and a control culture by running it through a filtering device with a 5 kDa cut-off. Running a western blot with these samples (see figure) we concluded that the immunotoxin was secreted as expected.

We can see distinctive molecular fragments in the immunotoxin lane which are not present in the negative control. The upper fragments (marked with red and purple arrows) seem to match the molecular size of the immunotoxin of about 39 kDa. For the shorter of the two fragments the export tag pelB may have been cut off. The smaller sized fragment of about 20 kDa in the whole cell lysate is most probably part of the degraded immunotoxin.

The band in the positive control (BH2, kDa) shows that the western blot worked. We see a clear band of the correct site (red arrow) which proves that the exportation works as expected and the immunotoxin is actually secreted. Obviously there is no need for a lysis device.

The same experiments were conducted for the proteins p25 and p28. No bands were detected on the western blots which leads us to the conclusion that these proteins were only very weakly expressed or not at all. This might be explained by the fact that we took the native sequence from Plasmodium falciparum . The genome of Plasmodium falciparum is very A-T-rich (put reference). We think that expression of p25 and p28 may be improved by codon optimizing it for expression in bacteria like E.Coli and Asaia like we did for the immunotoxin. Additional to the Western Blots we tried purifying the proteins using Ni-NTA columns. This still needs futher improvement.

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