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

Protein Expression and Purification
Fengliang JinDexiang Gu

Abstract

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. O...Continue Reading

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Citations

May 19, 2009·Applied Microbiology and Biotechnology·Aiping WangJunping Wang
Nov 24, 2011·Bioprocess and Biosystems Engineering·Xueyan RenXiaodong Zheng
Oct 12, 2012·World Journal of Microbiology & Biotechnology·Mingfu NiuPuyan Chen
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Sep 29, 2007·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Li WangYi Pang
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Feb 16, 2007·Protein Expression and Purification·Xiaoxia XuWenqing Zhang
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Feb 11, 2015·Experimental and Therapeutic Medicine·Mingfu NiuPeng Wan
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Dec 12, 2012·Experimental and Therapeutic Medicine·Chunhe GuoHouquan Jiang
Jun 26, 2020·Frontiers in Bioengineering and Biotechnology·Baseer AhmadRijun Zhang

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