Rasmussen-Wilson S J, Palas J S, Wolf V J, Taft C S, Selitrennikoff C P
MycoTox, Inc., Denver, Colorado 80262, USA.
Appl Environ Microbiol. 1997 Sep;63(9):3488-93. doi: 10.1128/aem.63.9.3488-3493.1997.
Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a protein in a Western blot of concentrated culture supernatants that comigrated with authentic zeamatin. Secreted zeamatin was active in inhibiting the growth of Candida albicans in an agar diffusion assay, indicating that zeamatin had been correctly synthesized, processed, and secreted by N. crassa.
植物基因的异源表达可能是生产植物蛋白的一种重要替代方法。我们研究了粗糙脉孢菌分泌玉米素（一种由玉米产生的蛋白质）的能力。将玉米素与粗糙脉孢菌产生的胞外水解酶糖化酶融合后可诱导其表达。在摇瓶液体培养的未转化粗糙脉孢菌中监测糖化酶的诱导及其他培养参数。通过将编码玉米素的cDNA插入包含粗糙脉孢菌糖化酶基因启动子、截短的开放阅读框和终止子序列的表达盒中构建了DNA质粒pGEZ。编码玉米素的cDNA在N端进行了修饰，以包含一个kex - 2蛋白酶位点，使嵌合产物在分泌途径中能够被切割。含有嵌合基因构建体的菌株在液体培养中生长，并诱导产生糖化酶和玉米素。玉米素抗体在浓缩培养上清液的蛋白质免疫印迹中检测到一种与天然玉米素迁移率相同的蛋白质。在琼脂扩散试验中，分泌的玉米素能够有效抑制白色念珠菌的生长，这表明粗糙脉孢菌能够正确合成、加工并分泌玉米素。
