Production and cytotoxicity against cancer cells of recombinant cecropin b-polyproline

Abstract

Cecropin B (CB) is a natural anticancer antimicrobial peptide which has the highest antibacterial activity among the Cecropin family. In this work, Cecropin B - polyproline (CB-PP) was constructed by adding polyproline (PP) which is a part of progesterone receptor (PR) to the C-terminus of CB sequence. The CB and CB-PP gene fragments were cloned into the pPICZαA expression vector. Then, the recombinant plasmid CB and CB-PP were transformed into Pichia pastoris strain KM71H by electroporation. The recombinant protein CB (rCB) and CB-PP (rCB-PP) were successfully produced by inducing with 0.5% methanol for 24 and 48 hours and purified using nickel column chromatography. The size of rCB and rCB-PP were found to be approximately 4.9 and 6.6 kDa, respectively. The cytotoxicity against six cell lines including human breast carcinoma (BT-474), human lung bronchus carcinoma (ChaGo-K1), human liver hepatocellular carcinoma (Hep-G2), human gastric carcinoma (Kato-III), human colon carcinoma (SW-620) and human lung fibroblast (WI-38) were investigated. All cell lines were treated with recombinant protein CB and CB-PP with the protein concentration between 0.078 µM – 20 µM for 72 hours. The results revealed that more than 50% of those five cancer cells were dead at the rage of 7 µM - 20 µM protein concentrations while more than 80% of normal cells was alive even treated with the same protein concentration. Those resultsmindicated that rCB และ rCB-PP could be produced by the transformed Pichia pastoris and the obtained recombinant proteins exhibited anticancer activity.