Bai Li, Hu Zhiming, Wang Fei, Xu Xiaoling, Xia Chang, Jin Liqin, Li Jinlong, Gao Jimin
Southern Medical University, Guangzhou, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2012 Oct;32(10):1389-93.
To obtain streptavidin-tagged human granulocyte-macrophage colony-stimulating factor (SA/hGM-CSF) fusion protein and evaluate its bioactivity .
PET24a-6His-SA-L-hGM-CSF and PET24a-hGM-CSF-L-SA-6His plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate the fusion proteins. The two fusion proteins were refolded by gradient dialysis after Ni-NTA affinity chromatography and finally purified using DEAE-sepharose FF anion exchange chromatography. MTT method was used to evaluate the effect of SA/hGM-CSF fusion proteins in inducing the proliferation of human erythroleukemia cells (TF-1). The efficiency of the fusion proteins for surface modification of biotinylated MB49 tumor cells was evaluated by flow cytometry.
The recombinant fusion proteins SA-hGM-CSF and hGM-CSF-SA were highly expressed in Rosetta (DE3) at about 20% of the total bacterial proteins, with a purity of about 96% after purification. The two fusion proteins exhibited bifunctional activities, namely the pro-proliferation effect on human erythroleukemia cells (TF-1) and SA-mediated high-affinity binding to biotinylated cell surfaces (with an anchoring modified rate of about 99%).
SA/hGM-CSF bi-fusion proteins obtained in this study lays the groundwork for the development of cancer cell vaccines with surface modification by hGM-CSF.
获得链霉亲和素标记的人粒细胞-巨噬细胞集落刺激因子（SA/hGM-CSF）融合蛋白并评估其生物活性。
构建PET24a-6His-SA-L-hGM-CSF和PET24a-hGM-CSF-L-SA-6His质粒，并在Rosetta (DE3)宿主菌中表达以产生融合蛋白。两种融合蛋白经Ni-NTA亲和层析后通过梯度透析复性，最后用DEAE-琼脂糖FF阴离子交换层析纯化。采用MTT法评估SA/hGM-CSF融合蛋白对人红白血病细胞（TF-1）增殖的诱导作用。通过流式细胞术评估融合蛋白对生物素化MB49肿瘤细胞表面修饰的效率。
重组融合蛋白SA-hGM-CSF和hGM-CSF-SA在Rosetta (DE3)中高表达，约占细菌总蛋白的20%，纯化后纯度约为96%。两种融合蛋白表现出双功能活性，即对人红白血病细胞（TF-1）的促增殖作用以及SA介导的与生物素化细胞表面的高亲和力结合（锚定修饰率约为99%）。
本研究获得的SA/hGM-CSF双融合蛋白为开发经hGM-CSF表面修饰的癌细胞疫苗奠定了基础。
