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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2020, Vol. 40 Issue (5): 40-47    DOI: 10.13523/j.cb.1912016
    
The Purification Procedure for the Recombinant HBcAg Virus-like Particle Easy to Generate Aggregation
XIE Hang-hang1,2,BAI Hong-mei1,2,YE Chao1,2,CHEN Yong-jun1,2,YUAN Ming-cui1,2,MA Yan-bing1,2,**()
1 Institute of Medical Biology,Chinese Academy of Medical Science&Peking Union Medical College,Kunming 650118, China
2 Yunnan Key Laboratory of Vaccine Research&Development on Severe Infectious Disease,Kunming 650118,China
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Abstract  

Objective: To explore an effective purification procedure of recombinant HBcAg virus like particles (VLPs) that are prone to aggregation. Methods: The expression of recombinant HBcAg protein was induced by IPTG in cultured E. coli. The centrifugation precipitates of bacteria after ultrasonic fragmentation were resuspended and dissolved in PBS buffer with different concentrations of urea. The VLPs behavior was analyzed and identified by density gradient centrifugation and electron microscopy. The precipitate solution was purified by Sepharose 4 FF gel filtration chromatography under the selected urea conditions, and the purified target protein was further desalinate to remove urea with PBS containing 30% sorbitol. The entire process was analyzed by SDS-PAGE and electron microscopy. Results: The ultrasonic precipitates resuspensed with PBS buffer containing 1mol/L urea was effective dissolve the aggregated VLPs, which showed the behavior of typical HBcAg VLPs in sucrose density gradient centrifugation, and morphology and structure of the particales were complete by electron microscope. VLPs were further purified after 1mol/L urea gel filtration. In the process of urea removal, PBS containing 30% sorbitol was used as the mobile phase, which effectively avoided the reaggregation of VLPs after urea removal. Conclusion: The combined application of urea and sorbitol provides an effective solution for the purification and preparation of VLPs with aggregation phenomenon.

Key words： Virus-like particle (VLP)      Purification      Aggregation      Urea      Sorbitol     
Received: 10 December 2019      Published: 02 June 2020
ZTFLH:  Q939.4  
Corresponding Authors: Yan-bing MA     E-mail: may@imbcams.com.cn
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Hang-hang XIE
Hong-mei BAI
Chao YE
Yong-jun CHEN
Ming-cui YUAN
Yan-bing MA
Cite this article:

XIE Hang-hang,BAI Hong-mei,YE Chao,CHEN Yong-jun,YUAN Ming-cui,MA Yan-bing. The Purification Procedure for the Recombinant HBcAg Virus-like Particle Easy to Generate Aggregation. China Biotechnology, 2020, 40(5): 40-47.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1912016     OR     https://manu60.magtech.com.cn/biotech/Y2020/V40/I5/40

Fig.1 The expression and identification of recombinant protein (a) The expressed recombinant protein aggregates in the ultrasonic precipitation M: Marker protein;1:Pre-induction; 2:Bacteria lysates of post-induction; 3:Ultrasonication supernatant; 4: Precipitation (b) Precipitation resuspended by PBS
Fig.2 The recombinant protein in the form of precipitation exists in the form of solubility in low concentration urea and is VLPs (a) SDS-PAGE analysis the resolved protein with different concentrations of urea (b) Sucrose density gradient ultracentrifugation M: Marker protein;1: Bacteria lysates of post-induction; 2-12:11 pipe solutions from top bottom (c) Electronic microscopy observation for VLPs after resolving with different concentrations of urea
溶解率和回收率 0.5mol/L 1mol/L 2mol/L 4mol/L
总沉淀(g) 0.162 0 0.162 0 0.162 0 0.162 0
剩余沉淀(g) 0.148 0 0.078 2 0.077 1 0.073 3
溶解沉淀(g) 0.014 0 0.083 8 0.084 9 0.088 7
沉淀溶解率(溶解沉淀/总沉淀×100%) 8.6% 51.7% 52.4% 54.8%
总蛋白质回收率[总蛋白质/(总蛋白质+沉淀蛋白质)×100%] 12.9% 80.2% 90.0% 92.1%
纯度(%) 41.5% 57.7% 55.9% 54.4%
目的蛋白回收率[总蛋白质×纯度/(总蛋白质+沉淀蛋白质) ×100%] 5.3% 46.3% 50.30% 50.10%
Table 1 Dissolution and protein recovery ratio of ultrasonic precipitation dissolved with different concentrations urea
Fig.3 The purification of recombinant protein by gel filtration chromatography (a) The representative picture of recombinant protein purified by gel filtration chromatography (b)SDS-PAGE analysis on the chromatography samples M: Marker protein;1: Bacteria lysates of post-induction;2: 1mol/L urea resuspended supernatant; 3: Peak 1; 4: Peak 2; 5: Peak 3
Fig.4 White recombinant protein dissolved with different protective agents (a)The recombinant protein urea-removed re-aggregates into a white precipitate M:Marker protein;1: White precipitate 2: Supernatant (b)30% sorbitol effectively prevents VLPs re-aggregation M:Marker protein; 1:10% sorbitol; 2:20% sorbitol; 3:30% sorbitol; 4:5% trehalose; 5:0.5mol/L glycine; 6:0.5mol/L NaCl;7:10% glycerin; 8:10% sucrose; 9:10% glucose
重组蛋白溶解率 10%
山梨醇		 20%
山梨醇		 30%
山梨醇		 5%
海藻糖		 0.5mol/L
甘氨酸		 0.5mol/L
NaCl		 10%
甘油		 10%
蔗糖		 10%
葡萄糖
总沉淀(g) 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0
剩余沉淀(g) 0.024 3 0.018 4 0.007 0.014 1 0.030 4 0.028 8 0.026 1 0.031 0 0.021 6
溶解沉淀(g) 0.005 7 0.012 6 0.023 0.015 9 0.000 6 0.001 2 0.004 9 0 0.008 4
溶解率(溶解沉淀/
总沉淀× 100%)		 18.4% 40.6% 74.2% 51.3% 1.9% 3.8% 15.8% 0% 27.1%
Table 2 Dissolution ratio of white recombinant protein dissolved with different protective agent
Fig.5 30% sorbitol buffer for removing urea with gel filtration M: Marker protein; 1: Bacteria lysates of post-induction; 2:Protein sample after removing urea with PBS containing 30% sorbitol as mobile phase buffer
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