Micheelsen Pernille Ollendorff, Ostergaard Peter Rahbek, Lange Lene, Skjøt Michael
Copenhagen Biocenter, Department of Molecular Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark.
J Biotechnol. 2008 Feb 29;133(4):424-32. doi: 10.1016/j.jbiotec.2007.11.012. Epub 2007 Dec 15.
An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants generated by gene replacement of the AOX1 gene was selected by PCR screening. Following methanol induction, expression levels reached 125 mgL(-1) from the wild-type coding region while expression from the two codon-optimized variants reached 65 and 125 mgL(-1), respectively. The protein was purified and characterized by Edman degradation, liquid chromatography mass spectrometry and insoluble blue starch assay, and was shown to possess the same characteristics as wild-type protein purified from barley grains.
利用甲醇诱导型醇氧化酶1（AOX1）启动子，在巴斯德毕赤酵母中构建了一个用于高水平表达天然大麦α-淀粉酶/枯草杆菌蛋白酶抑制剂（BASI）的表达系统。为优化表达，设计了两个密码子优化的编码区，并与野生型编码区一起表达。为确保天然成熟蛋白的分泌，使用了来自酿酒酵母的α交配因子分泌信号的截短版本。为了能够比较不同克隆的表达水平，通过PCR筛选选择了通过AOX1基因置换产生的单插入转化体。甲醇诱导后，野生型编码区的表达水平达到125 mgL(-1)，而两个密码子优化变体的表达水平分别达到65和125 mgL(-1)。该蛋白经埃德曼降解、液相色谱质谱分析和不溶性蓝色淀粉测定法进行纯化和表征，结果表明其具有与从大麦籽粒中纯化的野生型蛋白相同的特性。
