Buensanteai Natthiya, Mukherjee Prasun K, Horwitz Benjamin A, Cheng Cheng, Dangott Lawrence J, Kenerley Charles M
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA.
Protein Expr Purif. 2010 Jul;72(1):131-8. doi: 10.1016/j.pep.2010.03.006. Epub 2010 Mar 15.
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.
有益真菌绿色木霉分泌一种富含半胱氨酸的小蛋白（Sm1），该蛋白可诱导双子叶植物和单子叶植物的防御反应，是角质层-悬铃木蛋白家族的成员。从绿色木霉中纯化Sm1会导致蛋白质产量较低，从而将该蛋白在作物病害防治中的应用限制在小规模试验中。为了提高Sm1的产量，我们将sm1基因克隆到pPIC9K载体中，用于转化巴斯德毕赤酵母菌株GS115的AOX1位点。基于PCR分析所示的载体插入片段的存在以及分泌高水平rSm1蛋白的能力，筛选出巴斯德毕赤酵母的转化子。确定了摇瓶中生产rSm1的最佳培养时间和甲醇浓度。在终浓度为1%的甲醇中培养4天后，估计一个毕赤酵母转化子可表达约55 mg/l的rSm1。分泌的rSm1通过硫酸铵沉淀、离子交换色谱和凝胶柱色谱进行纯化。SDS-PAGE和Western印迹分析表明，毕赤酵母中表达的纯化rSm1可被抗Sm1多克隆抗体识别。通过对rSm1胰蛋白酶消化产物的ESI/MS/MS分析验证了蛋白质序列。获得了大于90%的肽段覆盖率，并确定与预测序列相同。MALDI/TOF/MS分析显示rSm1的分子量为13.1 kDa，高于天然Sm1（12.6 kDa）。对纯化蛋白的Edman测序显示N端有六个氨基酸的延伸（EAEAYV）。这种延伸是由于Ste13蛋白酶活性不足，无法有效切割Kex2切割位点下游的间隔区（EAEA）所致。用rSm1或天然Sm1处理的玉米（品种银皇后）表现出两个防御基因的诱导。这种激发子产量的提高对特种作物的病害防治具有重要意义。
