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| The Study on Fermentation and Purification Process of HBsAg Binding Protein(SBP) in Pichia pastoris |
| DONG Yue, SU Cai-xia, YANG Yan, ZHU Nai-shuo |
| State Key Lab of Genetic Engineering, Institute of Biomedical Science, School of Life Sciences, Fudan University, Shanghai 200433, China |
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Abstract The HBsAg binding protein (SBP) was screened out from human liver cDNA phage expression library with HBsAg as a probe. SBP has the ability to enhance the immune response of Hepatitis B vaccine. It is proved to be a potential efficient immune adjuvant. The secretory SBP-expressing Pichia pastoris strains with high protein expression quantity has successfully constructed, then was fermented in the 18L fermentor. The results showed that SBP could achieve the maximum expression after 38h in methanol induction according to real time detecting; meanwhile hybrid proteins were the least so that it's the most appropriate time to collect bacteria liquid.15L supernatant could be obtained at low temperature centrifugation. Through the ultrafiltration membrane package whose interception was 5kDa, the supernatant was concentrated to about 2L. Then the concentrated fermentation supernatant was passed through sephadex S200 and the TDEAE column respectively. The attained 300ml SBP protein was with a purity of 98% and 1.125mg/ml as concentration. The SBP yield from fermented liquid was about 22.5mg/L.Finally, the lyophilized proteins were preserved at low temperature. The work on the fermentation process optimized the time, identified the purification process and lay a solid foundation for the mass production of SBP.
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Received: 02 September 2014
Published: 25 December 2014
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