Heterologous Expression Of Human Antimicrobial Peptide LL-37 In Pichia Pastoris

Antimicrobial peptides are an important component of the innate immune system of most living organisms against invading pathogens. LL37 is the only cathelicidin-derived antimicrobial peptide found in humans. Not only do they possess an antibacterial, antifungal and antiviral function, they also show a chemotactic effect, neutralizing endotoxin and regulation of apoptosis. The purpose of this paper is to build a heterologous expression of human antimicrobial peptide LL37 in Pichia pastoris.Firstly, we constructed secretion expression strain GS115-9K-LL37. The cells were dead after induced by methanol for 10 h. The antimicrobial activity was very high measured by inhibition zone. This work mainly aims to decrease the inhibition of antimicrobial peptide on yeast and enhance the production.(1) We designed a fermentation and membrane filtration coupled system. However, no product was detected by filtration every 6 h and every 10 h. Thus the cultivation-filtration coupled system did not work for LL-37 fermentation (2) Expressing antibacterial peptide LL37 in peroxisome may space apart the membrane and other intracellular organelles, thereby avoiding the toxic of antimicrobial peptides on cells. We constructed a strain GS115-3.5K-LL37-SKL expression in peroxisomes. Antimicrobial activity could be detected after 36 h induction in 5-L bioreactor, but the yield was not high. So another strain GS115-3.5K-LL37-GFP-SKL was constructed. The GFP fluorescence intensities of cells and broth during the cell wall breaking process was measured by microplate reader. There was only about 19.6% of the green fluorescent protein in supernatant. (3) Tandem repeat multimers of LL37 was designed and secretion expression strains containing different copies of LL37 gene was constructed. However strains could not grow normally when starting methanol induction. We speculate that tandem multimers protein was also harmful to the strains. (4) To decrease the inhibition of antimicrobial peptide on yeast. Two strains, GS115-9K-GFP-EN-LL37-HIS6 and GS115-9K-LL37-DP-GFP-HIS6 were constructed and screened for different copy number by G418. It seemed the fusion proteins expressed by high copy number strains greatly inhibited the strain growth. However, the fusion protein expressed by low copy number strains showed mild inhibitive effect on cell growth. Proteins expressed by low copy number strains were then purified by nickel column affinity chromatography. The western blot analysis showed that the proteins was positive.