摘要
目的:包涵体在变复性前通常需要用洗涤液多次重复洗涤以除去杂质,这种洗涤方式往往造成包涵体收率低或洗涤时间长或纯度不高,直接影响到重组蛋白产品最终的产量和质量。通过对sTNFR1包涵体的洗涤条件进行优化,以期指导该品种的工业化生产。方法:首先采用由脱氧胆酸钠和尿素组成的两因素五水平的析因设计,以洗涤后目的蛋白的含量为评价指标,进行方差分析,选出最优组合;通过对方差结果的分析提出不同于重复洗涤的分步洗涤方式,选出较优的分步洗涤方式;然后放大洗涤样品量,验证重复洗涤和分步洗涤的结果;另外,针对不同发酵批次和产量的样品,进一步验证比较重复洗涤和分步洗涤的效果。结果:重复洗涤的方差结果表明单独使用脱氧胆酸钠或尿素洗涤的产量优于两者的联合使用,但其纯度低于两者的联合使用,其中1mol/L尿素洗涤3次(W3组合)获得的目的产量最高;分步洗涤中2%脱氧胆酸钠洗涤第一次,2%脱氧胆酸钠+2mol/L尿素洗涤第二次的洗涤方式(FW6组合)既能有效提高目的蛋白纯度,又节约了时间,是较优的组合方式;同一发酵水平(约20%目的产量)不同规模样品(3g和50g)分别经过W3和FW6洗涤后,蛋白纯度提高到约26%和31%,且后者的目的收率高于前者,表明分步洗涤优于重复洗涤;针对不同发酵水平的样品(约10%和60%目的产量),分步洗涤的效果也优于重复洗涤,但对于低发酵水平的样品,分步洗涤对其纯度的提高更明显。结论:通过对该包涵体洗涤条件的摸索,找到一种较重复洗涤更有效的分步洗涤方法,既提高了目的纯度又节约了洗涤时间,为包涵体的洗涤提供了一种新的思路。
Objective ： Overexpression of recombinant protein in prokaryotic cell often results in the formation of aggregates called inclusion bodies （IBs）. IBs need to be washed repeatedly before denaturing to remove impurities with one washing buffer, but it often result in low yield, long time, or low purity, which directly affect the yield and quality of the recombinant protein. By optimizing the washing condition of sTNFR1 IBs, its industrial production can be guided. Methods： First, screening one better way from the 25 repeat-washing buffers which composed of two factors sodium deoxycholate and urea, and five levels in each factor, and the yield of target protein is as the evaluation index. Second, according to the ANOVA results of repeat-washing, a new step- washing method is proposed, which is different from the repeat-washing method. In addition, the above two different washing methods be compared by enlarging sample amount and different batches of fermentation. Results： ANOVA results of repeat-washing method showed that sodium deoxycholate or urea alone is superior to both, and W3 （lmol/L Urea wash 3 times） obtain the highest yield of target protein. In step-washing method, FW6（2% sodium deoxycholate is used first and 2% sodium deoxycholate ＋ 2mol/L Urea is used in the second step） can effectively improve the purity of target protein and save time. The same batch of fermentation （ about 20% target protein） of different scale sample （3g and 50g） washed by W3 and FW6, purity of target protein was increased to 26% and 31% respectively, and the later target protein yield is higher than the former. The results indicated that step-washing method is better than repeat-washing method. For the different level of fermentation sample （about 10% and 60% target protein ）, step-washing is better than repeat-washing and step-washing method can obviously improve the target protein purity for the low level of fermentation sample. Conclusion： A step-washing method which is more effective than the r
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2013年第9期45-52,共8页
China Biotechnology
基金
"十二五"重大新药创制科技重大专项课题资助项目(2011ZX09506-007)
关键词
包涵体
脱氧胆酸钠
尿素
Inclusion bodies（IBs） Sodium deoxycholate Urea
