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| Study on Washing Method of Recombinant sTNFR1IBs |
| LUO Li1, Qin2, Jiao-rong2 |
1. Sichuan Industrial Institute of Antibiotics, Chengdu 610052, China;
2. Chengdu Institute of Biological Products Co., Ltd., Chengdu 610023, China |
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Abstract 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(1mol/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 repeat-washing method is proposed by the optimization of IBs washing conditions, which improves the purity of target protein, saves the washing time, but also provides a new idea for IBs washing.
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Received: 10 May 2013
Published: 25 September 2013
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