Expression of recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) in Pichia pastoris: purification and characterization.

Hybrid antibacterial peptide CA-MA (cecropinA(1-8)-magainin2(1-12)) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach of expression of hybrid peptide CA-MA in methylotrophic yeast, Pichia pastoris, the gene of CA-MA was obtained by recursive PCR (rPCR) and cloned into the vector pPICZalpha-A. The SalI-linearized plasmid pPICZalpha-CA-MA was transformed into P. pastoris SMD1168 by electroporation. The expression was induced for 96h with 1.0% methanol at 28 degrees C, pH 5.0. Recombinant CA-MA was purified by reversed-phase HPLC and 22 mg pure active CA-MA was obtained from 1L fermentation culture. Tricine-SDS-PAGE indicated that recombinant CA-MA protein molecular weight is 2.6 kDa. Mass spectrometry of purified CA-MA demonstrated a single large signal for the molecular ion [M+2H+](2+) at 1281.07 m/z, identical to that of the putative protein (2.56 kDa). Antimicrobial assays showed that CA-MA has a broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. This is the first report on the heterologous expression of a hybrid antibacterial peptide with molecular weight below 3.0 kDa in P. pastoris. Our results demonstrate that functional CA-MA can be produced in sufficient quantities using P. pastoris for use in further studies on functionality and diagnostic applications.

Expression and characterization of a housefly cecropin gene in the methylotrophic yeast, Pichia pastoris.

A 371 bp full-length cDNA (GenBank Accession No. DQ232774) was obtained from housefly Musca domestica by using degenerate primers and subsequent amplification by 5'- and 3'-RACE. The cecropin gene, Mdcec and Mdcec/6His, was cloned into expression pPICZalpha-A vector and was expressed in the methylotrophic yeast, Pichia pastoris. The recombinant Mdcec was purified using cationic exchange chromatography and 1.2mg pure active Mdcec was obtained from 100ml culture broth supernatant. To facilitate purification of Mdcec, the C-terminal 6His-tagged Mdcec was also expressed in P. pastoris. The recombinant Mdcec/6His was purified to homogeneity by a nickel chelating sepharose column and 2.0mg pure active Mdcec/6His was obtained from 100ml culture broth supernatant. Anti-microbial assays demonstrated that Mdcec had broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. Mdcec/6His showed a similar activity to Mdcec against bacteria, but a slight higher activity against fungi. These results indicate that the 6His-tag can enhance the cationic nature and stability of Mdcec. This is the first report on the heterologous expression of a cecropin and cecropin with a 6His tag in P. pastoris. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional M. domestica cecropin for both research and industrial purpose.

[Correspondence analysis between occurrence type of Dendrolimus punctatus and topographical and vegetative features].

The relationship between the occurrence type of Dendrolimus punctatus and the geographical and vegetative features in subcompartments was analyzed using correspondence analysis and shown by a figure of cluster. The results showed that the majority subcompartments in which D. punctatus occurred frequently were at low elevation and low slope, and distributed among low hills or middle hills, while the subcompartments at high elevation or high slopes, or in low mountains or middle mountains were safe-type. The subcompartments with only one dominant species, especially Pinus elliottii, were easily attacked by D. punctatus. The subcompartments in conifer and broadleaf mixed forest had less insect pests than those in conifer mixed forest.