WorkBeads系列介质与C4介质层析纯化汉逊酵母表达的HBsAg效果的比较

Purification of HBsAg expressed in Hansenula polymorpha by hydrophobic interaction chromatography with WorkBeads series and C4 media

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 purification efficacy of HBsAg expressed in Hansenula polymorpha by hydrophobic interaction chromatography using columns 3146VL, 3147L, 3148H and 3149VH with various butyl densities in WorkBeads 40/100013 Bus low series of Bio-Work and CIM Monoliths C4 of BIA. Methods HBsAg was dissolved with various loading buffers, then loaded onto the column pre-filled with WorkBeads type 4 and C4 butyl chromatographic column, and eluated with various elution buffers. The flow- through and elution peaks were collected and determined for HBsAg content, based on which the flow-through and elution rates were calculated, and the optimal loading and elution buffers were screened. Results The optimal column for purification of HBsAg was 3147L, while the optimal loading buffer was 20 mmol/L PB containing 4% ammonium sulfate. Neither 20 mmol/L PB nor 20 mmol/L PB containing 30% isopropanol was the ideal elution buffer. The optimal loading buffer on CIM Monoliths C4 was 50 mmol/L MOPS containing 3% ammonium sulfate and 20 mmol/L PB containing 4% ammonium sulfate, while the optimal elution buffer was 50 mmol/L MOPS containing 0. 1% Triton X-100. Conclusion It is feasible to introduce WorkBeads series which may be used repeatedly and CIM Monoliths C4 which is resistant to high flow rate into the purification of HBsAg expressed in Hansenulapolymorpha.