Osborne L C, Georgiades J A, Johnson H M
Infect Immun. 1979 Jan;23(1):80-6. doi: 10.1128/iai.23.1.80-86.1979.
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.
通过使用T细胞促有丝分裂剂葡萄球菌肠毒素A，在小鼠脾细胞的转瓶培养中进行了高滴度（10(2.2)至10(4)U/ml）免疫干扰素（II型）的大规模生产。用55%至80%饱和度的硫酸铵沉淀90%的这种干扰素，可使其浓缩20倍并纯化2至6倍。将这种干扰素应用于牛血清白蛋白（BSA）-Affi-Gel 10柱或羟基磷灰石柱后，可回收100%的干扰素活性。通过BSA-Affi-Gel 10柱层析，7%的回收活性未结合，45%用pH梯度5至7洗脱，48%用1M NaCl洗脱。与起始材料相比，从BSA-Affi-Gel 10柱洗脱的pH和盐洗脱干扰素分别纯化了62倍和390倍。对从BSA-Affi-Gel 10柱洗脱的pH和盐洗脱干扰素峰进行再层析，并未改变其洗脱模式。用不同pH和盐含量的缓冲液从BSA-Affi-Gel 10柱逐步洗脱干扰素，也可实现大于300倍的纯化。通过从BSA-Affi-Gel 10柱的任何一种洗脱程序，均可获得每毫克蛋白质高达2×10(5)U干扰素的比活性。通过羟基磷灰石柱层析，5%的回收活性未结合，20%用盐梯度洗脱，75%用30%甘油洗脱。这两个馏分的纯化倍数分别为107倍和16倍。干扰素的Ultrogel AcA 34柱层析产生了两个活性峰，一个主要峰的分子量约为40,000，一个次要峰的分子量为70,000至90,000。因此，通过不同类型的层析发现免疫干扰素是异质性的。
