Cloning & Studies of Neurospora Alkaline Protease

我们用两个启动子--GRG-1的葡萄糖抑制启动子和B-微管蛋白的构成启动子--证明了异源蛋白在粗糙脉孢菌中的表达。表达牛前凝乳酶原基因的神经孢子菌转化子可在培养基中积累具有酶活性的可溶性成熟凝乳酶。异源蛋白表达系统的一个重要因素是合适的宿主菌株，而对于这种宿主菌株来说，缺乏蛋白酶的突变株往往更具优势。脉孢子菌在缺乏碳、氮或硫的情况下，在培养液中有蛋白质存在的情况下会分泌一种碱性蛋白酶。当诱导时，碱性蛋白酶占分泌蛋白的40%。我们正在克隆野油菜分泌型碱性蛋白水解酶的编码基因和基因组位点，以开发一种改进的宿主和额外的表达载体来表达异源蛋白。用于表达异源蛋白质的微生物系统的发展使得能够分离出更多数量稀缺的蛋白质，通常比从原始来源获得更高的纯度。这样表达的蛋白质具有广泛的应用，从工业用途到蛋白质生物化学研究，再到人类治疗。这些克隆将被用来创造碱性蛋白酶缺失的干扰突变，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.