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A cDNA library was constructed from the mycelium of Trichoderma asperellum T4 and a highly expressed gene fragment named EplT4 was found.In order to find a more efficient and cost-effective way of obtaining EplT4, we attempted to produce EplT4 by using a Pichia pastoris expression system.The gene encoding EplT4, with an additional 6-His tag at the C-terminus, was cloned into the yeast vector pPIC9K and expressed in the P.pastoris strain GS 115.Transformants of P.pastoriswere selected by PCR analysis and the ability to secrete high levels of the Eplt4 protein was determined.The optimal conditions for induction were assayed in shake flasks using an enzyme-linked immunosorbent assay (ELISA).The yield of purified EplT4 was approximately 20 mg/L by nickel affinity chromatography and gel filtration chromatography.Western blot and MALDI/TOF/MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometer) analysis revealed that the recombinant EplT4 was expressed in both monomers and dimers.Soybean leaves treated with the monomer demonstrated the induction of four defense genes.Early cellular events in plant defense response were also observed after incubation with EplT4.Soybean leaves protected by EplT4 against Cercosporidium sofinum Hara indicated that EplT4 produced in P.pastoris was biologically active and would be potentially useful for practical applications.