Bockhorn Jessica, Balar Bharvi, He Dongming, Seitomer Eden, Copeland Paul R, Kinzy Terri Goss
Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA.
Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17682-7. doi: 10.1073/pnas.0805642105. Epub 2008 Nov 11.
Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.
硒代蛋氨酸（SeMet）是一种具有潜在毒性的氨基酸，但它在制备用于X射线晶体学中多波长反常色散或单波长反常色散相位分析的标记蛋白质方面却是一种有价值的工具。目前尚不完全清楚高浓度SeMet在真核细胞中发挥毒性作用的机制。利用酿酒酵母制备用于X射线晶体学的完全取代的SeMet蛋白质的尝试一直受到限制。我们对酿酒酵母可行的单倍体无效等位基因菌株库进行了抗SeMet筛选。编码胱硫醚γ-裂解酶的CYS3基因缺失导致对SeMet的抗性最高。此外，SSN2基因缺失导致对SeMet的抗性增加以及Cys3p水平降低。缺乏CYS3的蛋氨酸营养缺陷型菌株能够在以SeMet作为唯一蛋氨酸来源的培养基中生长，在总蛋白质中实现基本100%的占有率。CYS3缺失菌株为在酵母以及可能的其他真核系统中制备SeMet取代蛋白提供了一种简便且经济高效的方法。
