Ezrin的原核表达及与其相互作用蛋白的研究

Study on prokaryotic expression of Ezrin and its interacting proteins

目的体外筛选人胰腺癌细胞中与Ezrin蛋白相互作用的蛋白。方法提取人胰腺癌Panc-1细胞总RNA,RT-PCR法扩增Ezrin基因。将扩增得到的Ezrin基因片段和pET15b质粒转化E.coli DH5α,获得pET15b-Ezrin重组质粒,双酶切后测序鉴定。将测序正确的重组质粒转化至E.coli BL21-CodonPlus(DE3)-RIL,以异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,对表达产物进行Ni2+-NTA亲和层析和分子筛纯化后,行SDS-PAGE、MALDI-TOF-MS/MS质谱分析和蛋白质印迹鉴定。裂解Panc-1细胞,获得总蛋白提取液,采用Pull-down方法体外筛选与Ezrin重组蛋白相互作用的蛋白,并对筛选出的目的蛋白进行双向凝胶电泳和MALDI-TOF-MS/MS质谱鉴定。结果RT-PCR扩增出的Ezrin基因片段(1761bp)与理论DNA表达片段大小一致,测序鉴定显示克隆的Ezrin基因序列与GenBank报道序列相符。SDS-PAGE、质谱分析和蛋白质印迹分析显示纯化后Ezrin重组蛋白相对分子质量为69400,与质谱分析的相对分子质量结果相符,为带有His-6标签的特异蛋白。双向凝胶电泳和质谱鉴定结果共筛选出可与Ezrin重组蛋白相互作用的7种蛋白,其分别为RNA-binding motif protein39(RBM39)、Transgelin(TAGLN)、Albumin(ALBU)、Formin-1(FMN1)、Rho GTPases(RHGBA)、Tetratricopeptide repeat protein16 (TTC16)、Syntaxin-binding protein1(STXBP1)。结论在大肠杆菌中成功表达、纯化了Ezrin蛋白,并在体外成功筛选出可信的与Ezrin蛋白相互作用的蛋白。

Objective To explore the Ezrin-interacting proteins of pancreatic cancer cells in vitro. Methods Total RNA of pancreatic cancer tissues was extracted for amplification of Ezrin gene by RT-PCR. The amplified Ezrin gene and the plasmid pET15b were transformed to the E.coli DHSct to construct a recombinant plasmid pET15b-Ezrin, and the correctly constructed pET15b-Ezrin was identified with restriction analysis and sequencing, pET15b-Ezrin was transformed to the E.coli BL21-CodonPlus （DE3） -RIL, and the expression of Ezrin was induced by IPTG. After being purified by Ni^2＋-NTA affinity column and molecular sieve, the expressive product was analyzed with SDS-PAGE, and MALDI-TOF-MS/MS, and then identified with Western blotting. Panc-1 cells were cracked to obtain total protein extract. The recombinant Ezrin-interacting proteins were isolated by Pull-down assay, and then identified with two-dimensional gel electrophoresis and MALDI-TOF-MS/MS. Results The Ezrin gene fragment at a length of 1761 bp was amplified by RT-PCR, which was consistent with expected. The sequence of amplified Ezrin gene was identical to that reported in GenBank. SDS-PAGE, MALDI-TOF-MS/MS, and Western-blotting showed the relative molecular weight of the recombinant Ezrin protein was 69 400, which was consistent with the consequence of MALDI-TOF-MS/MS, and the protein was his-tagged specific protein. Identified by two-dimensional gel electrophoresis and MALDI-TOF-MS/MS, seven recombinant Ezrin-interacting proteins were RNA-binding motif protein39 （RBM39）, Transgelin （TAGLN）, Albumin （ALBU）, Formin-1 （FMN1）, Rho GTPases （RHGBA）, Tetratricopeptide repeat protein 16 （TrC16）, and Syntaxin-binding protein 1 （STXBP1）, respectively.Conclusion Ezrin protein was successfully expressed in E.coli and purified, and the creditable Ezrin-interacting proteins were successfully identified in vitro.