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Phylogeny Analysis, Multi-copy Expression and Characterization of Fungi Defensin--plectasin

Author: ZhangJun
Tutor: WangJianHua
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: Defensin Mycelium neomycin Bioinformatics Multicopy Expression Antibacterial properties Stability
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 99
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Abstract

The mycelium ADM is isolated from the growth in the North Ousong Lin surface saprophytic ascomycetes Pseudoplectania nigrella, a fungal defensin. Mycelium neomycin efficient anti Gram-positive bacteria and no cell toxicity, very new drug's potential anti-Gram-positive bacterial infections. In this study, by the mycelium the neomycin protein sequence BLAST in UniProtKB (ExPasy) database analysis found 62 kinds of homology with mycelium neomycin defensins. The phylogenetic tree constructed defensins found that 11 of the same evolutionary branch mycelium neomycin. The amino acid sequence analysis found 62 defensin sequences in addition to the conserved cysteine, the Loop glycine; α-helical structure, both proline and iso-leucine; the C end highly conserved lysine same. 11 and mycelium neomycin in the same evolutionary branch sequence hydrophobic cluster analysis found clustered hydrophobic amino acids at the C-terminus; positively charged surface analysis showed that defensins in the evolutionary process of the charged surface exist select; mycelium neomycin Bioinformatics analysis helps mycelium neomycin expression host strain and the choice of expression. In accordance with the yeast codon usage preference optimize the mycelium neomycin gene sequence and cloned into the expression vector pPICZαA to get recombinant expression vector pPICPlectasin of recombinant expression in Pichia pastoris X-33. High density fermentation, a secreted protein of 748.63μg mL-1, including restructuring mycelium neomycin content of 71.79% of the total secreted proteins. Using purified recombinant mycelium neomycin of Sephadex G-25. The product was purified by RP-HPLC separation, mass spectrometry determination of the molecular weight of the recombinant mycelium neomycin 4404.256 Da, with its theoretical value (4404.82 Da) match. Study attempts to build on the basis of expression of single copy mycelium neomycin, the mycelium neomycin series of multiple copies of the expression vector, using pPICZαA on a pair of isocaudamer BglII and BamHI digestion sites, a single copy of mycelial neomycin expression vector digestion, the resulting mycelium neomycin expression cassette with the linear vector series, built from the mycelium neomycin two Copy, four copies, and eight-copy expression vectors, transformed yeast cells, quantitative PCR copy number verification of transformants. Each copy transformants induced expression, to find hyphae neomycin single copy and four-copy transformants copy number and expression level there is a linear relationship. To antibacterial spectrum measured reorganization mycelium neomycin found mycelium neomycin Gram-positive Staphylococcus aureus (Staphyloccocus aureus ATCC 25923), Staphylococcus epidermidis (Staphyloccocus epidermidis ATCC 26069), Streptococcus pneumoniae (Streptococcus pneumoniae CVCC 2350) and pig streptococcus (Streptococcus suis CVCC 3309) has antibacterial activity, minimum inhibitory concentration (MIC) of the Streptococcus suis (S. suis CVCC 3309) 2μg mL-1, and Gram-negative bacteria on bacteria wire neomycin is not sensitive; hemolytic experiments found the mycelium neomycin rabbit blood erythrocytes with hemolytic; pH stability experiments found that recombinant mycelium neomycin in a buffer solution of pH 2.0-10.0, treatment did not affect its anti-Staphylococcus aureus activity; thermal stability was found that the activity of all of the anti-Staphylococcus aureus mycelium neomycin can be maintained within the range of 30-80oC, and even processed under the conditions of 100oC for one hour, was able to maintain 50% activity; anti protease found that recombinant mycelium neomycin excellent resistance to degradation ability of pepsin and papain, but sensitive to trypsin degradation experiments. In summary, this study by the mycelium neomycin bioinformatics analysis to clarify the choice of the charged surface defensin evolutionary process role, and the successful realization of the high-level expression in Pichia pastoris of mycelium neomycin, while establishing a A method to build to improve the expression level of target gene expression cassette series body. Reorganization mycelium neomycin antibacterial properties, and its application to lay the foundation.

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