Large-scale production and partial purification of mouse immune interferon.
Large-scale production and partial purification of mouse immune interferon.
复制标题
小鼠免疫干扰素的规模化生产及部分纯化。
作者：
L. Osborne;J. Georgiades;H. Johnson
使用T细胞有丝分裂原葡萄球菌肠毒素A，在小鼠脾细胞的滚筒培养物中大规模生产高滴度（10（2.2）至10（4）U/ml）免疫干扰素（II型）。用55至80％饱和硫酸铵沉淀90％的干扰素，导致20倍浓缩和2至6倍纯化。将该干扰素应用于牛血清白蛋白 (BSA)-Affi-Gel 10 或羟基磷灰石柱后，100% 恢复了干扰素活性。通过 BSA-Affi-Gel 10 色谱法，7% 的回收活性未结合，45% 用 pH 梯度 5 至 7 洗脱，48% 用 1 M NaCl 洗脱。与起始材料相比，从 BSA-Affi-Gel 10 柱中 pH 洗脱和盐洗脱的干扰素分别纯化了 62 倍和 390 倍。对 BSA-Affi-Gel 10 柱的 pH 洗脱干扰素峰和盐洗脱干扰素峰进行重色谱分析，未改变其洗脱模式。使用不同 pH 值和盐含量的缓冲液从 BSA-Affi-Gel 10 柱中逐步洗脱干扰素也可实现超过 300 倍的纯化。使用 BSA-Affi-Gel 10 柱的任一洗脱程序均可获得每毫克蛋白质高达 2 x 10(5) U 干扰素的比活性。通过羟基磷灰石色谱法，回收的活性中5％未结合，20％用盐梯度洗脱，75％用30％甘油洗脱。两个级分的纯化分别为 107 倍和 16 倍。干扰素的 Ultrogel AcA 34 色谱法产生两个活性峰，一个主峰的分子量约为 40,000，一个次峰的分子量为 70,000 至 90,000。因此，通过不同类型的色谱分析，发现免疫干扰素具有异质性。
Large-scale production of high-titered (10(2.2) to 10(4) U/ml) immune interferon (type II) was carried out in roller cultures of mouse spleen cells by using the T-cell mitogen staphylococcal enterotoxin A. Precipitation of 90% of this interferon by 55 to 80% saturated ammonium sulfate resulted in a 20-fold concentration and a two- to sixfold purification. After application of this interferon to either bovine serum albumin (BSA)-Affi-Gel 10 or hydroxylapatite columns, 100% of the interferon activity was recovered. By BSA-Affi-Gel 10 chromatography, 7% of the recovered activity was not bound, 45% was eluted with pH gradient 5 to 7, and 48% was eluted with 1 M NaCl. The pH- and salt-eluted interferons from the BSA-Affi-Gel 10 column were purified 62- and 390-fold, respectively, when compared with the starting materials. Rechromatography of the pH- and salt-eluted interferon peaks from the BSA-Affi-Gel 10 column did not alter their elution patterns. Stepwise elution of interferon from the BSA-Affi-Gel 10 columns with buffers of various pH and salt contents also resulted in greater than 300-fold purification. Specific activities of up to 2 x 10(5) U of interferon per mg of protein were attained with either elution procedure from BSA-Affi-Gel 10 columns. By hydroxylapatite chromatography, 5% of the recovered activity was not bound, 20% was eluted with a salt gradient, and 75% was eluted with 30% glycerin. Purification was 107- and 16-fold, respectively, for the two fractions. Ultrogel AcA 34 chromatography of the interferon resulted in two peaks of activity, a major one with a molecular weight of approximately 40,000 and a minor peak of molecular weight 70,000 to 90,000. Thus, by different types of chromatography, immune interferon was found to be heterogeneous.