ErbB2受体酪氨酸激酶激酶区的表达与纯化

Expression and Purification of Receptor Tyrosine Kinase ErbB2 Kinase Domain

摘要:

以GFP融合表达的形式在毕赤酵母中表达具有生物活性的受体酪氨酸激酶ErbB2的激酶区。构建受体酪氨酸激酶激酶区与GFP的融合表达载体pPIC3.5K, 转化毕赤酵母GS115, 通过组氨酸营养缺陷型筛选, G418高拷贝菌株筛选, 以及摇瓶诱导表达筛选, 选取较高水平表达菌株进行5升罐培养, 以镍亲和层析手段纯化得到蛋白表达产物, 进行SDS-PAGE分析和酶联免疫反应检测酶活。结果表明在毕赤酵母中成功诱导表达了约100 kD的激酶融合蛋白并具有激酶活性。该研究为筛选ErbB2的抑制剂奠定了基础。

Abstract:

The kinase domain of receptor tyrosine kinase(RTK) ErbB2 was expressed fused with GFP in Pichia pastoris. Recombinant expression vector pPIC3.5K was constructed in Escherichia coli TOP10. The right P. pastoris transformants were screened on his-deficient plates and YPD-G418 plates by turns after electroporation of recombinant vector, and then induced by methanol in baffled shake bottles. The strain with highest protein yield was scaled up in a 5 L fermentor. Recombinant protein was analyzed with tyrosine kinase assay after Ni2+ affinity chromatograph. Results showed that the 100 kD recombinant protein with tyrosine kinase activity was successfully expressed in P. pastoris.