Recombinant production of therapeutic peptide tilapia piscidin 4 (TP4) in pichia pastoris expression system and investigation of its effect on HT-29 colorectal cancer cells

Tilapia piscidin 4 (TP4), a cationic peptide with high antimicrobial activity, is part of the immune system of Nile Tilapia (Oreochromis niloticus). In addition to its antimicrobial properties, it has various biological significances, including wound healing and anticancer properties. In the scope of this thesis, the codon-optimized sequence of the TP4 peptide was expressed in the Pichia pastoris KM71H expression system under the pPICZαA expression vector, which includes the strong and inducible AOX1 promoter. Recombinant TP4 peptide was purified by Ni-NTA affinity chromatography and its antibacterial activity was confirmed by MIC and MBC methods. TP4 was applied to HT-29 colon cancer and HGF-1 healthy gingival fibroblast cells and cytotoxic effects on the cells were investigated by MTT, TAS-TOS, MDA methods and the expression levels of genes (BAX, BCL-2, CASP3) and proteins involved in apoptosis were investigated by RT-qPCR and ELISA methods. In this thesis, it was shown for the first time that recombinant TP4 has strong anticancer activity in human HT-29 cell line.