Expression of truncated tobacco osmotin in Escherichia coli: purification and antifungal activity

• Published in: Biotechnology letters 2011-03, Vol.33 (3), p.539-543
• Main Authors: Tzou, Ywh-Min, Huang, Tung-Shi, Huggins, Kevin W, Chin, Bryan A, Simonne, Amarat H, Singh, Narendra K
• Format: Article
• Language: English
• Subjects: Anti-yeast activity; Antifungal activity; Antifungal Agents - metabolism; Antifungal Agents - pharmacology; Antimicrobial agents; Applied Microbiology; Beads; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Candida albicans; Candida albicans - drug effects; Cellulose; Cobalt; Cryptococcus neoformans; Cryptococcus neoformans - drug effects; Culture; E coli; Electrophoresis, Polyacrylamide Gel; Elution; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Gene expression; Inclusions; Life Sciences; Microbiology; Nicotiana - genetics; Nicotiana - metabolism; Original Research Paper; Osmotin; Pichia - drug effects; Pichia methanolica; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Proteins - pharmacology; Proteins; Recombinant; Recombinant tobacco osmotin; Saccharomyces cerevisiae; Saccharomyces cerevisiae - drug effects; Strain; Tobacco; Urea; Yeasts
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-010-0453-z

Purpose of work Tobacco osmotin is a functional homolog of mammalian adiponectin, and has antifungal activity. This work was undertaken to produce recombinant osmotin that has previously been unsuccessful because of its toxicity. Expression of recombinant tobacco osmotin (rOSM) in Escherichia coli inclusion bodies has been achieved. The optimal pH for rOSM expression in ZYM 505 medium is 7.0 at OD₆₅₀ of 1.5 of culture growth. The rOSM from the inclusion body was extracted with 8 M urea, and purified using CM-cellulose and cobalt-agarose bead affinity chromatography to a high purity. Approximately 80% of the rOSM remained bound to CM-cellulose and Cobalt-agarose beads after initial elution. The yield of purified rOSM was between 40 and 50 mg from 2 l of culture. Repeated elution of protein from CM-cellulose and Co-agarose increased the yield of rOSM to 200 mg from 2 l culture. The purified rOSM showed variable antifungal activities against two pathogenic yeast strains; Cryptococcus neoformans, Candida albicans, and non-pathogenic strains; Saccharomyces cerevisiae and Pichia methanolica.