重组人角质细胞生长因子在毕赤酵母中的表达、纯化及活性测定

Expression,purification and activity detemination of recombinant human keratinocyte growth factor in Pichia pastoris

目的:利用巴斯德毕赤酵母(Pichia pastoris)重组表达人角质细胞生长因子(human keratinocyte growth factor,hKGF),为实现规模化生产重组hKGF奠定基础。方法:根据已报道的hKGF成熟肽序列,人工合成由毕赤酵母偏好密码子组成的编码hKGF的DNA片段后连接到分泌型表达质粒pPICZαA中,获得重组表达质粒并转化毕赤酵母X-33中进行表达。优化发酵条件,并利用5 L生物反应器进行中试发酵。发酵上清通过超滤及层析方法分离纯化重组蛋白,并利用MMT法检测其对恒河猴肺上皮细胞的促增殖活性。结果:在20℃,甲醇诱导60 h后,获得分子量约为28 kD的目的蛋白,表达量约占菌体上清总蛋白的14.1%。发酵液经肝素亲和层析和Sephardex G-25分子筛分离获得纯度为95.0%以上的重组蛋白,得率为12 mg/L。该重组hKGF具有糖基化修饰,能显著促进恒河猴肺上皮细胞增殖,其ED50约为57μg/L。结论:利用毕赤酵母成功表达出糖基化修饰及具促恒河猴肺上皮细胞增殖活性的重组hKGF。

To determine the conditions of recombinant human keratinocyte growth factor （hKGF） ex- pression for large - scale production using Pichia pastoris （ P. pastoris）. METHODS ： The DNA fragment encoding hKGF mature peptide was artificial synthesized. This artificial gene, which consisted of 20 codons of preference in P. pastoris, was cloned into secretory expression vector pPICZotA and transformed into P. pastoris X - 33 strain. Under the optimal con- ditions in a 5 L fermentor, the target protein was purified using the combination of uhrafiltration and chromatography. The pro - proliferative activity of this protein for rhesus monkey lung epitelial cell line 4MBr - 5 was detected by MTI assay. RESULTS ： After 60 h of fermentation induced by methanol at 20℃, the expression level of recombinant hKGF was up to 14.1% of the total supernatant proteins. Subsequent filtration through heparin affinity chromatography and Sephadex G - 25 yielded 12 mg/L target protein with purity 〉 95.0%. The glycosylated hKGF stimulated the proliferation of 4MBr -5 cells in vitro and its EDs0 was 57 μg/L. CONCLUSION： The glycosylated hKGF was successfully expressed using P. pastoris and was able to improve the proliferation of 4MBr- 5 cells in vitro.