Method for separating and purifying recombined hepatitis b surface antigen expressed by Hansenula yeast
A technology of hepatitis B surface antigen and Hansenula yeast, applied in the field of protein separation and purification, can solve the problems of low activity recovery rate, many HBsAg steps, long cycle, etc., and achieve the effects of simple production process, great practical application value and short operation cycle.
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Embodiment 1
[0035] Example 1. Laboratory-scale separation and purification experiment 1
[0036] Take 200ml of the culture supernatant of the recombinant hepatitis B surface antigen expressed by Hansenula yeast cultured by conventional fermentation, centrifuge and wash the Hansenula yeast cells expressing the hepatitis B surface antigen, then place it in a high-pressure homogenizer and add The weight ratio of the liquid to be disrupted is 0.05% Triton X-100 and 1 mM PMSF, and the disruption is repeated 6 times, and the cell disruption rate reaches 90%.
[0037] Centrifuge the broken cells at 20000g for 20 minutes to remove cell debris. Adjust the pH of the supernatant to 5.5 with 1.0M NaOH and 1.0M HCl, and the conductivity is less than 5.0mS / cm, and then feed it to Q SepharoseFF ion exchange In the chromatography column (GE Healthcare, 30cm×15cm ID), the column volume (CV) is 2000ml, and the flow rate is 1.0ml / (h·ml gel). The chromatographic process is followed by buffer I (20mM phosphate buff...
Embodiment 2
[0041] Example 2: Laboratory-scale separation and purification experiment 2
[0042] Take 200ml of the culture supernatant of the recombinant hepatitis B surface antigen expressed by Hansenula yeast cultured by conventional fermentation, centrifuge and wash the Hansenula yeast cells expressing the hepatitis B surface antigen, then place it on a ball mill, and add the crushing liquid The weight ratio of 0.3% Triton X-100 and 0.1 mM PMSF was repeated twice, and the cell disruption rate reached 50%.
[0043] Centrifuge the broken cells at 6000g for 30min to remove cell debris. Adjust the pH of the supernatant to 8.5 with 1.0M NaOH and 1.0M HCl, and the conductivity is less than 20mS / cm, and then feed it to the Q SepharoseFF ion exchange layer In the analysis column (GE Healthcare, 30cm×15cm ID), the column volume (CV) is 2000ml, and the flow rate is 20ml / (h·ml gel). The chromatographic process is followed by buffer I (20mM Tris-HCl buffer, pH 8.5) balance, feed, buffer I rebalance, 0...
Embodiment 3
[0047] Example 3. Laboratory-scale separation and purification experiment 3
[0048] Take 50ml of the culture supernatant of the recombinant hepatitis B surface antigen expressed by Hansenula yeast cultured by conventional fermentation, centrifuge and wash the Hansenula yeast cells expressing the hepatitis B surface antigen, then place it on a high-pressure homogenizer, and add the The weight ratio of the disrupted liquid was 0.1% Triton X-100 and 10 mM PMSF, and the disruption was repeated 3 times, and the cell disruption rate reached 70%.
[0049] Centrifuge the broken cells at 10000g for 20min to remove cell debris. Adjust the pH of the supernatant to 7.0 with 1.0M NaOH and 1.0M HCl, and the conductivity is less than 10mS / cm, and then feed it to the DEAE QZTFF ion exchange layer In the analysis column (National Biochemical Engineering Center, 30cm×5.5cm ID), the column volume (CV) is 500ml, and the flow rate is 10ml / (h·ml gel). The chromatographic process is followed by buffer ...
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