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.
Jin
Fengliang
F
State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen Zhongshan University, Guangzhou 510275, PR China.
Xu
Xiaoxia
X
Zhang
Wenqing
W
Gu
Dexiang
D
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006793:Houseflies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000432:Methanol / Q000378:metabolism*; D008969:Molecular Sequence Data; D010641:Phenotype; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D019476:Insect Proteins; D011994:Recombinant Proteins; D000432:Methanol