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Expression of magainin-2 in Saccharomyces cerevisiae
Magainin-2是一種具有抗細菌、黴菌、寄生蟲、病毒、腫瘤及避孕功能之抗菌蛋白，為magainin家族中重要的一員。存在於非洲青蛙(Xenopus laevis)皮膚中。由23個胺基酸所組成，為雙極性之α-helix結構。抗菌機制目前研究推測與在目標細胞細胞膜上形成離子通道有關。目前已經有許多magainin之衍生物已經進入第三期臨床研究階段(phase III)，相信不久的將來將可應用在治療疾病上，是一個具有前瞻性之抗菌蛋白。因此，許多科學家紛紛嘗試以各種表現系統來表現magainin以達到量產之目的。而本實驗將以Saccharomyces cerevisiae為宿主建構表現系統來表現magainin-2。將含有magainin-2 核酸片段之表現載體，轉形至S. cerevisiae 表現宿主，再以含有半乳糖之選擇性培養液誘導表現。產物經SDS-PAGE、抗菌活性分析後，證實為一具有抗菌活性之物質。為了確認表現產物，將利用高性能液相層析法及質譜層析法作進一步的分析。
Magainin-2, an antimicrobial peptide belongs to magainin family, was first isolated from the skin of Africa frog ( Xenopus laevis ) by Zasloff. It exhibits potent antimicrobial activities against a broad range of microorganisms, including bacteria, parasites, fungi, and viruses. In addition to its antimicrobial activities, magainin-2 also contains contraceptive activities. It consists of 23 amino-acid residues with an amphipathic α- helix conformation. The mechanism of its actions may be due to ion channel formation in cell membrane. To date, current data implicate that magainin derivatives have been in the development of the phase III clinical study. It is believed that magainin-2 is a potential antimicrobial peptide for medical applications in the future. In order to the produce magainin-2 in large quantities, different expression systems have been developed. In this experiment, I used Saccharomyces cerevisae for magainin-2 production. The DNA fragment encoding magainin-2 was cloned into expression vector and transformed into S. cerevisae host cell. Finally, gene expression was induced by galactose selection broth. The succeseful expression was proved by SDS-PAGE and antibacterial assay. We will use HPLC and mass spectra metry to further study the structure of our products.
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