Clare J J, Rayment F B, Ballantine S P, Sreekrishna K, Romanos M A
Department of Molecular Biology, Wellcome Biotech, Beckenham, Kent, UK.
Biotechnology (N Y). 1991 May;9(5):455-60. doi: 10.1038/nbt0591-455.
We have used the methylotrophic yeast, Pichia pastoris, to express high levels of tetanus toxin fragment C, a potential subunit vaccine against tetanus. In high biomass fermentations fragment C was induced to 27% of total cell protein or about 12 g/l of culture. The purified protein was as effective as native fragment C in immunizing mice. In order to optimize fragment C production, we have examined the parameters affecting foreign gene expression in Pichia. The level of expression was found to be largely independent of the site of chromosomal integration of the gene (AOX1 or HIS4), the type of integrant (insertion or transplacement), and the methanol utilisation phenotype of the host strain (Mut+ or Muts). The most important factor in obtaining high levels was the presence of multiple integrated copies of the fragment C expression cassette. Multicopy clones could be isolated from transformations using DNA fragments targeted for single-copy transplacement into the chromosome. These multicopy transformants were surprisingly stable over multiple generations during growth and induction in high cell density fermentations. Analysis of chromosomal DNA from these clones suggests that they arose by circularization of the transforming DNA fragment in vivo followed by multiple insertion into the chromosome via repeated single crossover recombination, in addition to the expected transplacement event. We have found this to be a general phenomenon and have used these multicopy "transplacement" clones to obtain high-level expression of several other foreign genes in Pichia.
我们利用甲基营养型酵母毕赤酵母来高效表达破伤风毒素C片段，它是一种潜在的破伤风亚单位疫苗。在高生物量发酵中，C片段的表达量诱导至占总细胞蛋白的27%，即每升培养物约12克。纯化后的蛋白在免疫小鼠方面与天然C片段一样有效。为了优化C片段的生产，我们研究了影响毕赤酵母中外源基因表达的参数。发现表达水平在很大程度上与基因在染色体上的整合位点（AOX1或HIS4）、整合体类型（插入或转座）以及宿主菌株的甲醇利用表型（Mut+或Muts）无关。获得高表达水平的最重要因素是C片段表达盒存在多个整合拷贝。多拷贝克隆可从使用靶向单拷贝转座到染色体中的DNA片段进行转化的实验中分离得到。在高细胞密度发酵的生长和诱导过程中，这些多拷贝转化体在多代中惊人地稳定。对这些克隆的染色体DNA分析表明，除了预期的转座事件外，它们是由体内转化DNA片段环化，随后通过重复的单交换重组多次插入染色体而产生的。我们发现这是一种普遍现象，并已利用这些多拷贝“转座”克隆在毕赤酵母中实现了其他几个外源基因的高水平表达。
