Cloning & Studies of Neurospora Alkaline Protease

我们利用两个启动子——葡萄糖抑制启动子grg-1和b -微管蛋白的组成启动子——证明了异源蛋白在粗神经孢子虫中的表达。表达牛凝乳酶前原cDNA的神经孢子虫在培养基中积累可溶的、酶活性的成熟凝乳酶。异种蛋白表达系统的一个重要因素是合适的宿主菌株，蛋白酶缺陷突变体通常是更好的选择。神经孢子菌在培养基中存在蛋白质时分泌碱性蛋白酶，同时也缺乏碳、氮或硫。在诱导时，碱性蛋白酶占分泌蛋白的40%。目前，我们正在研究克隆稻棘草分泌碱性蛋白酶的cDNA和基因组位点，以开发一种改良的宿主和额外的表达载体，用于表达外源蛋白。用于表达异源蛋白的微生物系统的发展使得分离出更多数量的稀缺蛋白成为可能，通常比从原始来源分离出的纯度更高。如此表达的蛋白质有广泛的应用，从工业用途，通过蛋白质生物化学研究，到人类治疗。这些克隆随后将用于制造碱性蛋白酶缺乏破坏突变，并将蛋白酶启动子和分泌信号肽添加到我们的表达载体库中。

We have demonstrated heterologous protein expression in Neurospora crassa using two promoters - the glucose repressible promoter of grg-1 and the constitutive promoter of the B-tubulin. Neurospora transformants expressing a bovine preprochymosin cDNA accumulate soluble, enzymatically active mature chymosin in the culture medium. One important element of heterologous protein expression systems is the appropriate host strain, for which protease deficient mutants are often superior. Neurospora secretes an alkaline protease in the presence of protein in the culture medium when also starved for carbon, nitrogen, or sulfur. When induced, the alkaline protease comprises 40% of secreted proteins. We are cloning cDNA and the genomic locus encoding the N. crassa secreted alkaline protease in order to develop an improved host and additional expression vectors for expression of heterologous proteins. %%% The development of microbial systems for expression of heterologous proteins has enabled the isolation of greater amounts of scarce proteins, often in greater purity than is possible from the original source. Proteins so expressed have applications ranging from industrial use, through protein biochemistry research, to human therapeutics. These clones will be used subsequently to create alkaline protease deficient disruption mutantions and to add the protease promoter and secretion signal peptide to our expression vector repertoire.