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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.
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Received: 10 December 2019
Published: 02 June 2020
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Corresponding Authors:
Yan-bing MA
E-mail: may@imbcams.com.cn
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