全长PTH(1-34)-HSA融合蛋白在毕赤酵母中的表达及其活性

Expression of intact PTH(1-34)-HSA fusion protein in Pichia pastoris and activity of expressed product

目的在毕赤酵母中表达全长甲状旁腺激素(Parathyroid hormone,PTH)(1-34)与人血清白蛋白(Human serumalbumn,HSA)的融合蛋白PTH(1-34)-HSA,并检测其活性。方法在质粒pPIC9K-PTH(1-34)2-HSA的基础上设计引物,引入6His标签及肠激酶酶切位点,从该质粒中扩增6His-EK-PTH(1-34)-HSA基因,插入pPIC9K载体中,构建重组表达质粒pPIC9K-6His-EK-PTH(1-34)-HSA,转化毕赤酵母GS115,甲醇诱导表达;表达的6His-PTH(1-34)-HSA融合蛋白通过两步亲和层析纯化和肠激酶酶切,获得全长的PTH(1-34)-HSA融合蛋白;检测融合蛋白对UAMS-32P成骨细胞增殖活力、碱性磷酸酶活性以及RANKL和OPG基因转录水平的影响。结果重组表达质粒经测序证实构建正确;表达的6His-PTH(1-34)-HSA融合蛋白相对分子质量约70 000,可与PTH抗体和HSA抗体特异性结合,发酵获得的融合蛋白的浓度为200 mg/L;纯化的融合蛋白可与抗PTH、HSA和6His标签抗体特异性结合,经肠激酶酶切后,该蛋白失去了与6His抗体反应的能力,保持了与PTH和HSA抗体结合的能力;酶切后的全长PTH(1-34)-HSA融合蛋白保持了PTH的生物活性,能促进UAMS-32P成骨细胞的增殖活力,提高细胞的碱性磷酸酶的活性,上调细胞RANKL基因的转录水平以及抑制OPG基因的转录水平。结论成功在毕赤酵母中表达了全长PTH(1-34)-HSA融合蛋白,有望提高PTH的成药性。

Objective To express the fusion protein of intact parathyroid hormone（PTH）（1-34） and human serum albumin（HSA） in Pichia pastoris and determine the activity of expressed product.Methods Primers were designed based on plasmid pPIC9K-PTH（1-34）2-HSA,in which a tag of 6His and a restriction site of enterokinase were introduced.6His-EK-PTH（1-34）-HSA gene was amplified from the plasmid and inserted into vector pPIC9K.The constructed recombinant plasmid pPIC9K-6His-EK-PTH（1-34）-HSA was transformed to P.pastoris GS115 for expression under induction of methanol.The expressed fusion protein 6His-PTH（1-34）-HSA fusion protein was purified by two-step affinity chromatography and digested with enterokinase,and the obtained intact PTH（1-34）-HSA was determined for effect on proliferation activity of osteoblasts UAMS-32P,activity of alkaline phosphatase as well as transcription levels of RANKL and OPG genes.Results Sequencing proved that recombinant plasmid pPIC9K-6His-EK-PTH（1-34）-HSA was constructed correctly.The expressed 6His-PTH（1-34）-HSA fusion protein,with a relative molecular mass of about 70 000,showed specific bindings to PTH and HSA antibodies.The concentration of fusion protein obtained by fermentation was 200 mg / L.Purified fusion protein showed specific bindings to antibodies against PTH,HSA and 6His tag.However,after digestion with enterokinase,the fusion protein lost its binding ability to the antibody against 6His tag,while those against PTH and HSA were maintained.The intact PTH（1-34）-HSA fusion protein maintained the biological activity of PTH,which promoted the proliferation activity of UAMS-32P cells,increased the activity of alkaline phosphatase,up-regulated the transcription of RANKL gene while inhibited that of OPG gene.Conclusion Intact PTH（1-34）-HSA fusion protein was successfully expressed in P.pastoris,which further indicated the possibility of PTH as a drug.