Wood M J, Komives E A
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0601, USA.
J Biomol NMR. 1999 Feb;13(2):149-59. doi: 10.1023/a:1008398313350.
Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P. pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P. pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P. pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10-100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N-labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment.
在毕赤酵母中的异源表达具有许多真核表达的优点，如正确折叠、二硫键形成、糖基化和分泌。与其他真核系统不同，毕赤酵母中的蛋白质生产在简单的基本限定培养基中进行，这使得该系统对于生产用于核磁共振分析的标记蛋白质具有吸引力。因此，对于那些无法从包涵体中复性或需要翻译后修饰以实现正确折叠或功能的蛋白质，毕赤酵母是核磁共振分析的首选表达系统。毕赤酵母中表达蛋白的产量严重依赖于生长条件，通过发酵达到高细胞密度已被证明可将蛋白质产量提高10至100倍。不幸的是，同位素富集发酵培养基成分的成本，尤其是15NH4OH，高得令人望而却步。我们报道了一些发酵方法，这些方法能够利用（15NH4）2SO4进行15N标记，利用13C-葡萄糖或13C-甘油对毕赤酵母中产生 的蛋白质进行13C标记。具有两个N-连接糖基化位点的凝血调节蛋白83个氨基酸片段的表达表明，对于同位素富集，发酵比摇瓶培养更具成本效益。
