Sasaki Yoshiharu, Miyoshi Daisuke, Sugimoto Naoki
Frontier Institute for Biomolecular Engineering Research FIBER, Konan University, Kobe, Japan.
Biotechnol J. 2006 Apr;1(4):440-6. doi: 10.1002/biot.200500032.
Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro.
活细胞含有高浓度的大分子，但几乎所有的实验生化数据都是从不能反映体内条件的稀溶液中获得的。为了了解生物分子在体内的行为，体外研究的性质被外推到体内条件；然而，活细胞内的分子条件本质上是拥挤的。本研究使用不同分子量的聚乙二醇（PEG）作为拥挤剂，研究分子拥挤对DNA聚合酶活性的影响。在各种条件下进行的聚合酶活性测定表明，T7和Taq DNA聚合酶的活性取决于拥挤剂的分子量和浓度。此外，平衡和动力学分析表明，随着PEG浓度的增加，聚合酶的结合亲和力增加，催化活性降低。基于聚合酶反应的定量参数，我们提高了分子拥挤条件下PCR扩增的效率。这些结果表明，生物分子结构和功能的定量测量对于理解生物分子在体内的行为以及体外生物技术应用是有用的。
