Expression of truncated tobacco osmotin in Escherichia coli: purification and antifungal activity.
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 OD(650) 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.
Department of Nutrition and Food Science, Auburn University, 201 Poultry Science Building, Auburn, AL 36849, USA.
Ywh-Min
YM
Tzou
Tung-Shi
TS
Huang
Kevin W
KW
Huggins
Bryan A
BA
Chin
Amarat H
AH
Simonne
Narendra K
NK
Singh
D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects; D003455:Cryptococcus neoformans / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000187:drug effects; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
D000935:Antifungal Agents; D010940:Plant Proteins; C073139:osmotin protein, Nicotiana tabacum