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Information × Registration Number 0208U006206, 0107U005244 , R & D reports Title Development of domestic producer of hepatitis B surface antigen as a vaccine component on the basis of methylotrophic yeasts popup.stage_title Head Sibirny Andriy Andriyovych, Registration Date 23-06-2008 Organization Institute of cell biology, National Academy of Sciences of Ukraine popup.description2 The vectors have been constructed, providing regulated PMOX HBsAg and human insulin precursor synthesis, multicopy integration and stability of integrated into producent genome expression cassettes, and selection of highly effective strains-producers in labarotory conditions. The HBsAg EAO2-H25 producent has been selected, carrying 4-5 copies of tandemly integrated рКО6 vector. We have shown that 0,5% Triton Х 100 in the cell lysis buffer іmproves HBsAg extraction. HBsAg expression regulation by different carbon sources in the EAO2-H25 strain has been tested in periodical culture. Examination of medium and carbon source effect on HBsAg synthesis revealed that sucrose and ethanol fully repress HBsAg synthesis in EAO2-H25, and menthanol and glucose provide approximately the same antigene production level, while xylose ensures the highest level of HBsAg expression. We have also demonstrated that methylamine is the best nitrogen source for HBsAg production among other analyzed (aspartate, urea), andprovides 30-40% increase in HBsAg synthesis comparing to the other traditional nitrogen source, ammonium sulfate. HBsAg production in EAO2-H25 strain in the xylose medium has been studied in fermetor's volume (1liter). The cells were pregrown in rich YPS medium to achieve high density in liquid culture, and then transferred to the medium supplemented with 1% xylose and methylamine for HBsAg synthesis induction. We have shown that in such conditions HBsAg percent makes up to 2,5% of total cell protein. However, for effective protein production in manufacture, the percent of HBsAg should be not less than 7-8. Since the medium composition and extraction method has already been optimized, the only method for HВsAg production increase is the increase in integrated expression cassette copies into the genome of producing strain. Product Description popup.authors popup.nrat_date 2020-04-02 Close