Expression and purification of biologically active Trichoderma virens proteinaceous elicitor Sm1 in Pichia pastoris

The beneficial fungus Trichoderma virens secretes a small cysteine-rich protein (Sm1) that induces defense responses in dicot and monocot plants and is a member of the cerato-platanin family. Purification of Sm1 from T. virens results in low protein yield limiting the application of this protein for crop disease protection to small-scale assays. To increase the yield of Sm1, we cloned the sm1 gene in the pPIC9K vector for transformation into the AOX1 locus of Pichia pastoris strain GS115. Transformants of P. pastoris were selected based on the presence of the vector insert as indicated by PCR analysis and the ability to secrete high levels of the rSm1 protein. The optimal incubation period and methanol concentrations for induction were determined for production of rSm1 in shake flasks. One Pichia transformant was estimated to express approximately 55 mg/l of rSm1 after 4 days culture in a 1% final concentration of methanol. The secreted rSm1 was purified by ammonium sulfate precipitation, ion exchange chromatography and gel column chromatography. SDS-PAGE and Western blot analysis revealed that the purified rSm1 expressed in Pichia was recognized by anti-Sm1 polyclonal antibody. The protein sequence was verified by ESI/MS/MS analysis of a tryptic digest of the rSm1. Greater than 90% peptide coverage was obtained and determined to be identical to the predicted sequence. The MALDI/TOF/MS analysis revealed the molecular mass of rSm1 to be 13.1 kDa, which is higher than native Sm1 (12.6 kDa). Edman sequencing of the purified protein revealed an N-terminal extension of six amino acids (EAEAYV). The extension is the result of insufficient activity of the Ste13 protease preventing efficient cleavage of the spacer (EAEA) downstream of the Kex2 cleavage site. Maize (cv. Silver Queen) treated with rSm1 or native Sm1 demonstrated the induction of two defense genes. Enhanced production of this elicitor has implications for the treatment of specialty crops to promote disease resistance.