Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, USA.
Department of Chemistry, University of Texas at Austin, Austin, Texas, USA.
Nat Biotechnol. 2018 Aug;36(7):624-631. doi: 10.1038/nbt.4154. Epub 2018 Jun 4.
Incorporation of the rare amino acid selenocysteine to form diselenide bonds can improve stability and function of synthetic peptide therapeutics. However, application of this approach to recombinant proteins has been hampered by heterogeneous incorporation, low selenoprotein yields, and poor fitness of bacterial producer strains. We report the evolution of recoded Escherichia coli strains with improved fitness that are superior hosts for recombinant selenoprotein production. We apply an engineered β-lactamase containing an essential diselenide bond to enforce selenocysteine dependence during continuous evolution of recoded E. coli strains. Evolved strains maintain an expanded genetic code indefinitely. We engineer a fluorescent reporter to quantify selenocysteine incorporation in vivo and show complete decoding of UAG codons as selenocysteine. Replacement of native, labile disulfide bonds in antibody fragments with diselenide bonds vastly improves resistance to reducing conditions. Highly seleno-competent bacterial strains enable industrial-scale selenoprotein expression and unique diselenide architecture, advancing our ability to customize the selenoproteome.
将稀有氨基酸硒代半胱氨酸掺入形成二硒键可以提高合成肽治疗剂的稳定性和功能。然而，这种方法在重组蛋白中的应用受到异质掺入、低硒蛋白产量和细菌生产菌株适应性差的阻碍。我们报告了经过改良的、适应性更好的重组硒蛋白生产的大肠杆菌菌株的进化。我们应用一种含有必需二硒键的工程化β-内酰胺酶，在连续进化的重组大肠杆菌菌株中强制依赖硒代半胱氨酸。进化后的菌株可以无限期地维持扩展的遗传密码。我们设计了一种荧光报告基因来定量体内硒代半胱氨酸的掺入，并表明 UAG 密码子完全被解码为硒代半胱氨酸。用二硒键替换抗体片段中原有的不稳定二硫键可大大提高其对还原条件的抗性。高度适应硒的细菌菌株能够实现工业规模的硒蛋白表达和独特的二硒键结构，提高了我们定制硒蛋白组的能力。
