大肠杆菌表达重组人生长激素的纯化

Purification of Recombinant Human Growth Hormone in E.coli

目的 :获得具有生物学活性的重组人生长激素 (rhGH)。方法 :PBV -GH/DH5α菌体经超声破菌、反复洗涤后获得包涵体。将包涵体变性、复性 ,用硫酸铵盐析 ,离子交换层析和凝胶层析进行纯化。产物经SDS -PAGE、HPLC、N末端 15个氨基酸序列检测验证。结果 :终产物rhGH纯度达 98.2 % ,比活性大于 3.0IU/mg。分子量为 2 2kDa ,N末端氨基酸序列与DNA序列推导的氨基酸序列完全一致。结论 :从自构建的PBV -GH/DH5α工程菌中获得高纯度、高活性重组人生长激素。其纯化工艺为中试生产提供可靠依据。

Recombinant human growth hormone (rhGH) engineering bacterial strain PBV-rhGH/DH5α was disrupted and washed to achieve expressed inclusion bodies. Then expressed inclusion bodies were denatured and renatured. rhGH was purified by salting out, ion exchange and sephacryl S-100 gel filtration chromatography. The purified protein was identified by SDS-PAGE, HPLC and sequencing analysis of 15 amino acids in N-terminal. The purity and specific activity of the purified rhGH reached 98% and 3.0 IU/mg respectively. The molecular mass was 22KD. N-terminal amino acid analysis was consistent with amino acid sequence based on DNA sequence. This research has laid solid foundation for pilot-scale production of rhGH.