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中国生物工程杂志

China Biotechnology
China Biotechnology  2011, Vol. 31 Issue (01): 46-50    DOI:
    
The Influence of Different Cell Disruption Methods on the Activity of the Extract in Cell-free Protein Synthesis System
WANG Xue1,2, QUAN Chun-shan3, WANG Jian-hua1,2, FAN Sheng-di3
1. Dalian Institute of Chemical Physica, Chinese Academy of Sciences, Dalian 116023, China;
2. Graduate University of Chinese Academy of Sciences, Beijing 100049, China;
3. Key Laboratory of Biochemical Engineering, State Ethnic Affairs Commission and Ministy of Education, Dalian Nationalities University, Dalian 116600, China
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Abstract  

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.



Key wordsCell-free protein synthesis system      G-6-PDH      Disruption method      Extract activity     
Received: 02 September 2010      Published: 25 January 2011
ZTFLH:  Q816  
Cite this article:

WANG Xue, QUAN Chun-shan, WANG Jian-hua, FAN Sheng-di. The Influence of Different Cell Disruption Methods on the Activity of the Extract in Cell-free Protein Synthesis System. China Biotechnology, 2011, 31(01): 46-50.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2011/V31/I01/46


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