Cloning & Studies of Neurospora Alkaline Protease

克隆

• 批准号: 9260436
• 负责人: John Marshall Ivy
• 金额: $ 5万
• 依托单位: Hawaii Biotechnology Group Inc
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1993-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9260436&HistoricalAwards=false
• 关键词: Cloning; & Studies; Neurospora; Alkaline

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.

我们使用两个启动子（grg-1 的葡萄糖抑制型启动子和 B-微管蛋白的组成型启动子）证明了粗糙脉孢菌中的异源蛋白表达。 表达牛前凝乳酶原 cDNA 的脉孢菌转化体在培养基中积累可溶性、具有酶活性的成熟凝乳酶。 异源蛋白质表达系统的一个重要因素是合适的宿主菌株，对于该宿主菌株，蛋白酶缺陷型突变体通常更优越。 当培养基中存在蛋白质且缺乏碳、氮或硫时，脉孢菌会分泌碱性蛋白酶。 当被诱导时，碱性蛋白酶包含 40% 的分泌蛋白。我们正在克隆编码粗糙脉孢菌分泌的碱性蛋白酶的 cDNA 和基因组位点，以开发改进的宿主和用于表达异源蛋白的额外表达载体。 %%% 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大量的稀有蛋白质，其纯度通常高于原始来源的纯度。 如此表达的蛋白质具有从工业用途、蛋白质生物化学研究到人类治疗的广泛应用。 这些克隆随后将用于创建碱性蛋白酶缺陷型破坏突变体，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。