|M.Sc Student||Mizrachi Roey|
|Subject||Expression of Antimicrobial Peptides from the Dermaseptin|
Family in Escherichia Coli by Gene Fusion
|Department||Department of Biotechnology and Food Engineering||Supervisors||Professor Amram Mor|
|Professor Yuval Shoham|
|Full Thesis text - in Hebrew|
The aim of this research was to express antimicrobial peptides from the dermaseptin family in microbial systems. Antimicrobial peptides constitute an important element of the unspecific branch of the immune system. They differ in size, shape, sequence, action range and in the manner they kill or neutralize target microorganisms. Due to their unique properties they are of interest to the pharmaceutical and food industries, therefore it is necessary to develop economical methods for large scale production.
Currently peptides are produced either by chemical synthetic strategies, which are speedy but at a high cost, or by using biological production in microbial cells, which is more cost-effective but highly toxic to the host. Therefore there is a need to neutralize their toxicity. One way to overcome this is by fusing the toxic peptide to an insoluble protein thus changing its spatial structure. This can also help the purification processes by formation of insoluble bodies. Another method used is fusion with an acidic fragment, which imitates natural biosynthesis.
In this work we chose to produce the peptide K 4K 20S4 of the dermaseptin S family. The first approach was to imitate its natural production course. We selected two acidic preserved sequences from the dermaseptins and magainin precursors. The fused acidic sequences did not contribute to lowering the toxicity of the peptide in presence of an inducer molecule and showed no ever-expression of the peptide.
The second approach involved fusing the peptide with one of three over expressed proteins: Lip65, XT6 and Reg. Even though three different proteins of diverse size, solubility and activity were tested, none has lowered the peptide’s toxicity, and their over-expression has repeatedly killed the producer bacteria.
Thirdly, we fused the peptide to a large protein along with an acidic fragment fused to the carboxylic terminal. The resulting fusion product was still highly toxic.
The method that did help reduce toxicity was a double fusion strategy where the peptide was intercalated between a protein the acidic fragment. The addition of the acidic fragment to the C-terminal managed to reduce its toxicity, yet only the XT6 fragment could also allow over-expression of the peptide.
In the future we hope to use this method to continue developing a system for bacterial expression of antimicrobial peptides in general, and of dermaseptin peptides in particular.