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目的比较WorkBeads 40/10000 Bus low系列3146VL、3147L、3148H、3149VH 4型不同丁基密度的疏水层析柱与BIA CIM Monoliths C4纯化汉逊酵母表达的HBsAg的效果。方法将HBsAg溶于不同的上样缓冲液中,分别上样于4型WorkBeads预装层析柱和C4丁基层析柱,用不同的洗脱缓冲液洗脱,分别收集上样流穿峰和洗脱峰,测定HBsAg含量,并计算HBsAg的流穿率和洗脱率,筛选最佳上样缓冲液和洗脱缓冲液。结果 3147L型柱最适于HBsAg纯化工艺,含4%硫酸铵的20 mmol/L PB是理想的上样缓冲液,20 mmol/L PB和含30%异丙醇的20 mmol/L PB均不是理想的洗脱缓冲液。CIMMonoliths C4最佳上样缓冲液是含3%硫酸铵的50 mmol/L MOPS和含4%硫酸铵的20 mmol/L PB;含0.1%Triton-100的50 mmol/L MOPS是最佳的洗脱缓冲液。结论可多次重复使用的WorkBeads系列介质和可耐受高流速的快速纯化介质CIMMonoliths C4均可用于汉逊酵母表达的HBsAg的纯化。
OBJECTIVE To compare the effect of WorkBeads 40/10000 Bus low series 3146VL, 3147L, 3148H, 3149VH 4 different butyl density hydrophobic columns with BIA CIM Monoliths C4 purified Hansenula HBsAg expression. Methods HBsAg was dissolved in different loading buffer and loaded on 4 types of WorkBeads prepacked column and C4 butyl chromatographic column separately. Different elution buffers were used to elute HBsAg. Elution peak, determination of HBsAg content, and calculate the HBsAg flowthrough rate and elution rate, screening the best sample buffer and elution buffer. Results The 3147L column was the most suitable for the HBsAg purification process. 20 mmol / L PB with 4% ammonium sulfate was the ideal loading buffer and neither 20 mmol / L PB nor 20 mmol / L PB with 30% isopropanol Ideal elution buffer. The optimal loading buffer for CIM Monoliths C4 was 50 mmol / L MOPS with 3% ammonium sulfate and 20 mmol / L PBB with 4% ammonium sulfate; 50 mmol / L MOPS with 0.1% Triton-100 was optimal Remove the buffer. CONCLUSIONS The WorkBeads series of media that can be reused repeatedly and CIMMonoliths C4, a fast-acting high-flow-resistant media, can be used for the purification of HBsAg expressed in Hansenula.