Cloning and High Level Production of Engineered Synthetic Cationic Antimicrobial Peptide using Methanol Inducible Pichia pastoris GS115

Abstract

Engineered synthetic cationic antimicrobial peptides are the potential alternative drugs to existing antibiotics. In the present study, a novel attempt for the intracellular production of engineered synthetic cationic antimicrobial peptide (escAMP) using Pichia pastoris was studied. The engineered synthetic cationic antimicrobial peptide gene was synthesized using overlapping PCR. An entirokinase and hydroxylamine hydrochloride cleavage sites are incorporated at N- and C- terminal end of escAMP respectively for easy purification. Later the gene was inserted into the MCS region of pPICZ-B vector. The synthetic peptide under the AOX1 promoter was integrated into the Pichia pastoris GS115 genome and the recombinant clones were screened by using antibiotic resistance. Expression profiles of recombinant peptide were done using glycerol and methanol based synthetic medium and analysed on 18 % Tricine-SDS-PAGE. Purification of the expressed peptide was done after cell disruption (10 cycles on time, 10 cycles off time and 10 min of total time) using 6X histidine tag followed by enzymatic cleavage. In this study, 67 gm of dry cell weight/L and 580 mg/L of purified escAMP was produced. The purified peptide is analysed for its anti microbial activity against different Gram positive and Gram negative microbes. For the first time smallest engineered synthetic cationic peptide was designed, cloned and expressed from methanol inducible Pichia pastoris GS115 and production ranges are encouraging.