Suzuki Motoo, Roy Rohini, Zheng Haiyan, Woychik Nancy, Inouye Masayori
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2006 Dec 8;281(49):37559-65. doi: 10.1074/jbc.M608806200. Epub 2006 Oct 4.
We developed a new bacterial expression system that utilizes a combination of attributes (low temperature, induction of an mRNA-specific endoribonuclease causing host cell growth arrest, and culture condensation) to facilitate stable, high level protein expression, almost 30% of total cellular protein, without background protein synthesis. With the use of an optimized vector, exponentially growing cultures could be condensed 40-fold without affecting protein yields, which lowered sample labeling costs to a few percent of the cost of a typical labeling experiment. Because the host cells were completely growth-arrested, toxic amino acids such as selenomethionine and fluorophenylalanine were efficiently incorporated into recombinant proteins in the absence of cytotoxicity. Therefore, this expression system using Escherichia coli as a bioreactor is especially well suited to structural genomics, large-scale protein expressions, and the production of cytotoxic proteins.
我们开发了一种新的细菌表达系统，该系统利用多种特性（低温、诱导导致宿主细胞生长停滞的mRNA特异性核糖核酸内切酶以及培养物浓缩）来促进稳定的高水平蛋白质表达，表达量可达总细胞蛋白的近30%，且无背景蛋白合成。使用优化载体，指数生长的培养物可浓缩40倍而不影响蛋白质产量，这将样品标记成本降低至典型标记实验成本的百分之几。由于宿主细胞完全生长停滞，在无细胞毒性的情况下，硒代甲硫氨酸和氟苯丙氨酸等有毒氨基酸能有效掺入重组蛋白中。因此，这种以大肠杆菌作为生物反应器的表达系统特别适用于结构基因组学、大规模蛋白质表达以及细胞毒性蛋白的生产。
