Zawada James, Swartz James
Department of Chemical Engineering, Stauffer III, Stanford University, Stanford, California, 94305-5025, USA.
Biotechnol Bioeng. 2006 Jul 5;94(4):618-24. doi: 10.1002/bit.20831.
Cell-free protein synthesis is a useful research tool and now stands poised to compete with in vivo expression for commercial production of proteins. However, both the extract preparation and protein synthesis procedures must be scaled up. A key challenge is producing the required amount of biomass that also results in highly active cell-free extracts. In this work, we show that the growth rate of the culture dramatically affects extract performance. Extracts prepared from cultures with a specific growth rate of 0.7/h or higher produced approximately 0.9 mg/mL of chloramphenicol acetyl transferase (CAT) in a batch reaction. In contrast, when the source culture growth rate was 0.3/h, the resulting extract produced only 0.5 mg/mL CAT. Examination of the ribosome content in the extracts revealed that the growth rate of the source cells strongly influenced the final ribosome concentration. Polysome analysis of cell-free protein synthesis reactions indicated that about 22% of the total 70S ribosomes are in polysomes for all extracts regardless of growth rate. Furthermore, the overall specific production from the 70S ribosomes is about 22 CAT proteins per ribosome over the course of the reaction in all cases. It appears that rapid culture growth rates are essential for producing a productive extract. However, growth rate does not seem to influence specific ribosome activity. Rather, the increase in extract productivity is a result of a higher ribosome concentration. These results are important for cell-free technology and also suggest an assay for intrinsic in vivo protein synthesis activity.
无细胞蛋白质合成是一种有用的研究工具，目前已准备好与体内表达竞争蛋白质的商业生产。然而，提取物制备和蛋白质合成程序都必须扩大规模。一个关键挑战是生产所需数量的生物质，同时还要得到高活性的无细胞提取物。在这项工作中，我们表明培养物的生长速率会显著影响提取物的性能。从特定生长速率为0.7/h或更高的培养物中制备的提取物，在分批反应中产生约0.9mg/mL的氯霉素乙酰转移酶（CAT）。相比之下，当源培养物生长速率为0.3/h时，所得提取物仅产生0.5mg/mL的CAT。对提取物中核糖体含量的检测表明，源细胞的生长速率强烈影响最终的核糖体浓度。对无细胞蛋白质合成反应的多核糖体分析表明，无论生长速率如何，所有提取物中约22%的70S核糖体存在于多核糖体中。此外，在所有情况下，70S核糖体在反应过程中的总体比产量约为每个核糖体22个CAT蛋白。看来快速的培养生长速率对于产生有生产力的提取物至关重要。然而，生长速率似乎并不影响核糖体的比活性。相反，提取物生产力的提高是核糖体浓度较高的结果。这些结果对于无细胞技术很重要，也提示了一种体内蛋白质合成内在活性的检测方法。
