Biomolecular Engineering Lab., School of Food and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan.
J Biosci Bioeng. 2012 Feb;113(2):154-9. doi: 10.1016/j.jbiosc.2011.10.007. Epub 2011 Nov 13.
Yeast's extracellular expression provides a cost-efficient means of producing industrially useful recombinant proteins. However, depending on the protein to be expressed, the production results in a poor yield, which is occasionally accompanied with loss of the expression plasmid and hence hampered growth of the host in the inducing medium. Here we propose an alternative approach, high cell-density expression, to improve the yield of a certain range of so-called difficult-to-express proteins. In this expression system, recombinant yeast cells resting in stationary phase (OD(660)=3-4) are suspended in a small aliquot of inducing medium to form a high cell-density culture (e.g., OD(660)=15). When applied to the yeast strains harboring Lentinula edodes laccase (Lcc1 or Lcc4) expressing plasmids, the high cell-density system allowed the host cells to synthesize elevated amounts of the laccase which resulted in >1000- to 6000-fold higher yield than those synthesized in a classical growth-associated manner. The resting cells required aerobic agitation for the maximum production. The production system also worked for other foreign enzymes but not for beta-galactosidase from Aspergillus oryzae or Escherichia coli, likely suggesting an involvement of chaperons that act on a certain range of secretory proteins.
酵母的细胞外表达为生产具有工业用途的重组蛋白提供了一种经济高效的方法。然而，根据要表达的蛋白质，生产结果产量低，偶尔伴随着表达质粒的丢失，从而阻碍了宿主在诱导培养基中的生长。在这里，我们提出了一种替代方法，即高密度表达，以提高某些所谓的难表达蛋白的产量。在这个表达系统中，处于静止期（OD（660）= 3-4）的重组酵母细胞悬浮在一小部分诱导培养基中，形成高细胞密度培养物（例如，OD（660）= 15）。当应用于携带香菇漆酶（Lcc1 或 Lcc4）表达质粒的酵母菌株时，高密度系统允许宿主细胞合成大量漆酶，其产量比经典生长相关方式合成的产量高出 1000 至 6000 倍。静止细胞需要有氧搅拌才能达到最大产量。该生产系统也适用于其他外源酶，但不适用于米曲霉或大肠杆菌的β-半乳糖苷酶，这可能表明伴侣蛋白的参与，伴侣蛋白作用于一定范围的分泌蛋白。
