重组人白细胞介素-11在毕赤酵母中的表达与纯化

[Expression and purification of recombinant human interleukin-11 in Pichia pastoris].

作者信息

Zhu J K, Xu Z X, Huang W D, Ming T S, Xie W, Xu Y, Zhang X G

机构信息

Department of Immunology, Suzhou University, Suzhou 215007, China.

出版信息

Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2001 Apr;23(2):127-31.

PMID:12905887

Abstract

OBJECTIVE

To express recombinant human interleukin-11 (rhIL-11) in methylotropic yeast Pichia pastoris.

METHODS

By designing and synthesizing an artificial gene for IL-11, the expression vector pPICZ alpha-A-IL-11 was constructed and introduced into Pichia pastoris by linearized electroporation. The rhIL-11 protein was identified by ELISA and SDS-PAGE analysis. The bioactivity was analyzed by B9-11 cell line. A combination of liquid chromatography was developed to purify the rhIL-11 from ferment supernatant.

RESULTS

The nucleotide sequence analysis indicated that the sequence of cloned artificial IL-11 gene accorded with that of designed; the secreted yield of rhIL-11 by yeast Pichia pastoris KM71-2424 in flask reached 60 mg/L. The biological activity of IL-11 in yeast supernatant and E. coli standard determined by B9-11 was 5.5 x 10(7) U/mg and 2.2 x 10(7) U/mg respectively. The rhIL-11 was purified to electrophoretic purity by a combination of liquid chromatography.

CONCLUSION

The human IL-11 artificial gene was obtained and successfully expressed in the Pichia pastoris(KM71-2424). The biological activity of IL-11 in yeast supernatant was significantly higher than that of E. coli standard. The rhIL-11 was purified to electrophoretic purity.

摘要

目的

在甲基营养型酵母毕赤酵母中表达重组人白细胞介素-11（rhIL-11）。

方法

通过设计和合成IL-11人工基因，构建表达载体pPICZα-A-IL-11，并通过线性化电穿孔法导入毕赤酵母。通过ELISA和SDS-PAGE分析鉴定rhIL-11蛋白。利用B9-11细胞系分析其生物活性。开发了一种液相色谱组合方法从发酵上清液中纯化rhIL-11。

结果

核苷酸序列分析表明，克隆的人工IL-11基因序列与设计序列一致；毕赤酵母KM71-2424在摇瓶中分泌rhIL-11的产量达到60mg/L。通过B9-11测定，酵母上清液中IL-11和大肠杆菌标准品的生物活性分别为5.5×10⁷U/mg和2.2×10⁷U/mg。通过液相色谱组合将rhIL-11纯化至电泳纯。