Heimo H, Palmu K, Suominen I
Department of Biochemistry and Food Chemistry, University of Turku, Finland.
Protein Expr Purif. 1997 Jun;10(1):70-9. doi: 10.1006/prep.1996.0713.
In this paper we report the expression in Pichia pastoris, purification, and characterization of the Aspergillus awamori glucoamylase catalytical domain (GAc). Pichia pastoris produced GAc to the level of 0.4 g per liter medium. This production level is about the same level as that gained for recombinant GA from Aspergillus and about 100-fold more than previously achieved by Saccharomyces cerevisiae. The GAc expressed in Pichia pastoris was purified by two independent chromatographic methods employing ion exchange or affinity chromatography to apparent homogeneity. The purified protein has a molecular weight of about 75,000 and specific activity of 78 units per milligram protein. The propeptide present in the glucoamylase N terminus was found to be removed correctly by P. pastoris. Glucoamylase produced by P. pastoris is N- and O-glycosylated, with 23% carbohydrate content. The N-linked oligosaccharides appear to be larger than in invertase, another glycoprotein heterologously expressed in P. pastoris. O-glycosides (studied to our knowledge for the first time in P. pastoris in this report) contribute about half of the total carbohydrate content in GAc. Purified GAc appears as multiple hands on isoelectric focusing with p1 values around 3.5, a value that is little higher than that for GAc produced in S. cerevisiae. GAc could be used as a versatile tool in studying protein expression in P. pastoris: as an affinity handle for other secreted proteins produced in P. pastoris, as a reporter gene when studying gene expression, and as a model protein in studying protein secretion and processing in P. pastoris.
在本文中，我们报道了泡盛曲霉糖化酶催化结构域（GAc）在毕赤酵母中的表达、纯化及特性。毕赤酵母每升培养基中产GAc的水平达0.4 g。该产量水平与来自曲霉的重组糖化酶所获产量水平大致相同，比酿酒酵母此前的产量高约100倍。通过采用离子交换或亲和色谱的两种独立色谱方法对毕赤酵母中表达的GAc进行纯化，使其达到表观均一性。纯化后的蛋白分子量约为75,000，比活性为每毫克蛋白78单位。发现毕赤酵母能正确切除糖化酶N端存在的前肽。毕赤酵母产生的糖化酶进行了N-糖基化和O-糖基化，碳水化合物含量为23%。N-连接寡糖似乎比在毕赤酵母中异源表达的另一种糖蛋白转化酶中的更大。O-糖苷（本报告中首次在毕赤酵母中进行研究）约占GAc总碳水化合物含量的一半。纯化后的GAc在等电聚焦时呈现多条带，pI值约为3.5，该值略高于酿酒酵母产生的GAc的pI值。GAc可作为研究毕赤酵母中蛋白质表达的通用工具：作为毕赤酵母中产生的其他分泌蛋白的亲和标签，作为研究基因表达时的报告基因，以及作为研究毕赤酵母中蛋白质分泌和加工的模型蛋白。
