Phylogeny Analysis, Multi-copy Expression And Characterization Of Fungi Defensin--plectasin

Plectasin is a defensin isolated from a fungus, Pseudoplectania nigrella, which is a black saprophytic ascomycete found on the floor of northern European pine forests. Plectasin exhibited strong antimicrobial activity against gram-positive bacteria and no cytotoxic to nomal cells. In this study, we search UniProtKB database in ExPasy by BLAST to find plectasin homologous sequence. We find 62 defensins that have homology with plectasin. 11 defensins were predicted by phylogenetic analysis of the 62 defensins, which have high homology with plectasin. By the sequence analysis, excluding the cystine, the glycine in Loop region, the praline and isoleucine in the head and tail ofα-helix and lysines in C- terminus were also shown high conservation in 62 defensins. By the hydrophobic cluster analysis (HCA), the hydrophobic amino acids were composed of a hydrophobic cluster at the C- terminus. The molecular modeling and the electrostatic surface plots suggested that the negatively charged amino acids were amidated at N-terminus, and resulting in the positive electrostatic surface was increased at total defensin molecule.The gene coding plectasin was optimized, synthesized and cloned into the expression vector pPICZαA. The recombinant plectasin with native N-terminus was successfully expressed in P. pastoris X-33. Following 120 h induction of a high density fermentation culture in P. pastoris, the amount of total secretion protein reached 748.63μg mL-1 and the yield of recombinant plectasin was 71.79% of the total secretion protein. The recombinant plectasin was purified by Sephadex G-25 column and RP-HPLC. The MALDI-TOF mass spectrum analysis revealed the molecular mass of recombinant plectasin to be 4404.256 Da, which is consistent with native plectasin (4404.82 Da). Based on the single copy, using the mothod of tandem expression cassette, take the BglII and BamHI as an isocaudarners, to construct mulit-copy recombinant expression vector of plectasin. The two-copy, four-copy, and eight-copy of pPICPlectasin were determined by Bgl II and BamHI. We have found a direct relationship between plectasin expression level in P. pastoris and the number of introduced plectasin genes.The recombinant plectasin exhibited strong antimicrobial activity against gram-positive bacteria S. aureus ATCC 25923, S. epidermidis ATCC 26069, S. pneumoniae CVCC 2350, and S. suis CVCC 3309. The MIC against S. suis CVCC 3309 for recombinant plectasin was 2μg mL-1. In addition, the recombinant plectasin had anti-S. aureus ATCC 25923 activities in a wide range of pH, from 2 to 10.0. Furthermore, it showed significant thermal stability and still possessed about 50% of initial activity after 100°C for 1 h. Moreover, the recombinant plectasin had high resistance to both papain and pepsin, but sensitive to trypsin.In conclusion, accordint to the mothod of bioinformatics we find that the selection of defensin at electrostatic surface during the evaluation. And this is the first report of recombinant expression plectasin with natural N-terminal in P. pastoris. At the same time, we established a method of progressive improve expression level by increasing the copy numbers of plectasin expression cassette in recombinant plasmid. In addition, the effects of pH value, temperature and protease on antimicrobial activity of plectasin suggest that plectasin might be an attractive candidate for development of traditional antibiotic substitute for feed usage.