不同细胞破碎方法对无细胞蛋白表达系统细胞抽提物活性的影响

The Influence of Different Cell Disruption Methods on the Activity of the Extract in Cell-free Protein Synthesis System

无细胞蛋白表达系统由于能够有效表达膜蛋白等有毒性蛋白,因此近二十年受到了关注,其蛋白表达产率有了显著的提高。细胞抽提物活性的高低是无细胞蛋白表达系统高效运行的关键,若找到简单易行的活性评估方法,将大大降低成本及时间。葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G-6-PDH)是糖代谢的戊糖磷酸途径中的关键调控酶,该酶可以被用来评估细胞抽提物的活性。以G-6-PDH的活性为指标对无细胞蛋白表达系统中的抽提物活性进行评价,并利用G-6-PDH活性评价体系对机械破碎、高压破碎以及超声破碎三种破碎方法进行了比较,得出了三种破碎方法的最佳破碎条件。机械破碎最佳破碎条件是5000r/min,用直径0.1 mm玻璃珠,破碎6次;高压破碎的最佳破碎压力为1 300bar;超声破碎最佳破碎条件是功率强度为总功率的60%,破碎30次。酶活性测定结果显示机械破碎和超声破碎得到的抽提物活性比高压破碎得到的抽提物活性略高。

Cell-free（CF） protein expression system can be effectively used for membrane protein and other toxic protein expression.In recent two decades,CF system has received wide range of attention and the yield was significantly improved.The extract activity is the critical factor that affects the efficiency of protein synthesis system.If a simple method could be established to evaluate the activity of the extract,lots of time and cost would be saved up.Glucose-6-phosphate dehydrogenase（G-6-PDH） was the key regulatory enzyme in the pentose phosphate pathway in the glucose metabolism,therefore the activity of G-6-PDH could be utilized as an indicator to assess the extract activity.A convenient and feasible method was established,which use the activity of G-6-PDH in the extract to evaluate the extract activity.Three kinds cell disruption methods were compared,which included mechanical crushing,high pressure crushing and sonication,and the optimum conditions of each method were obtained.The mechanical crushing method got the most active extract by using 0.1mm-diameter glass beads at 5 000r/min for 6 times.When the disruption pressure was 1 300 bar,the activity of the extract reached the highest point.The sonication method got the best result at 60% power level,30 cycles.The results indicated that the activity of the extract obtained by mechanical crushing and sonication methods were a little higher than the high pressure disruption.