Cloning and Expression of a New Gene Encoding an Antifungal Protein in Phytolacca americana

protein (Pa-AFP) with molecular weight about 4 kD was purified from the seeds of Phytolacca americana L. , which obviously inhibits the growth of Rhizoctonia solani Kiihn in vitro. The authors isolated mRNA from the seeds of pokeberry and designed a degenerate PCR primer according to the N-terminal sequence of the purified protein. The full-length cDNA encoding Pa-AFP was cloned by RT-PCR and 5'-RACE and sequenced. The deduced amino acid sequence indicates that a preprotein with 65 amino acid residues is firstly translated and then processed to a mature protein with 38 amino acids. The DNA encoding the mature protein was subcloned into expression vector pGEX-4T1, and expressed efficiently in E. coli BL21 as a GST- Pa-AFP fusion protein. The fusion protein was purified by glutathione-Sepharose 4B affinity colmnn chromatography. The purified fusion protein was specifically digested by thrombin and the Pa-AFP was further purified by filtration column chromatography.