Solubilization and renaturation of biologically active human bone morphogenetic protein-4 from inclusion bodies.
Solubilization and renaturation of biologically active human bone morphogenetic protein-4 from inclusion bodies.
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DOI：
10.1016/j.btre.2018.e00249
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发表时间：
2018-06
期刊：
影响因子：
--
通讯作者：
Pepelanova I
中科院分区：
文献类型：
--
作者：
Gieseler GM;Ekramzadeh K;Nölle V;Malysheva S;Kempf H;Beutel S;Zweigerdt R;Martin U;Rinas U;Scheper T;Pepelanova I
在原核宿主中以包涵体形式表达具有生物活性的重组人骨形态发生蛋白-4。对不同的复性配方进行了试验，以获得最佳的二聚产率。阳离子交换膜层析法一步纯化二聚体。该产物可诱导诱导多能干细胞分化为滋养层细胞。商品化细胞培养的重组人骨形态发生蛋白-4与大肠杆菌产物的比较。在原核宿主中成功表达了具有生物活性的人骨形成蛋白-4(hBMP-4)。为此，在大肠杆菌中克隆了hBMP-4基因，并以非活性聚集态的形式表达该蛋白。洗涤和增溶后，用快速稀释法进行重组人骨形成蛋白4单体的体外复性。本研究考察了不同复性条件对重组人骨形态发生蛋白-4二聚体的影响。二聚过程对pH、蛋白质浓度和聚集抑制物的存在非常敏感。相比之下，氧化还原条件和离子强度并没有像预期的那样影响复性。用阳离子交换膜色谱一步将二聚体与其余单体、聚集体和宿主细胞污染物分离。在大肠杆菌中表达的重组人骨形成蛋白-4二聚体具有生物学活性，能够诱导滋养层细胞分化和诱导人多能干细胞(HPSCs)形成原始条纹。
Biologically active rhBMP-4 was produced in a prokaryotic host as inclusion bodies. Different refolding recipes were tested for optimal dimerization yield. One-step purification of dimer with cation-exchange membrane chromatography. The product induces trophoblast differentiation in induced pluripotent stem cells. Comparison between commercial rhBMP-4 from cell culture and product from E. coli. Biologically active human bone morphogenetic protein-4 (hBMP-4) was successfully produced in a prokaryotic host. For this aim, hBMP-4 cDNA was cloned in Escherichia coli (E. coli) and the protein was produced in a non-active aggregated form. After washing and solubilization, in vitro refolding of the rhBMP-4 monomer was performed using rapid dilution. In this study, different refolding conditions were tested for the dimerization of rhBMP-4 by one-factor-at-a-time variation. The dimerization process was found to be sensitive to pH, protein concentration and the presence of aggregation suppressors. In contrast, redox conditions and ionic strength did not impact refolding as expected. The dimer was separated from the remaining monomer, aggregates and host cell contaminants in a single step using cation-exchange membrane chromatography. The rhBMP-4 dimer produced in E. coli was biologically active as demonstrated by its capability to induce trophoblast differentiation and primitive streak induction of human pluripotent stem cells (hPSCs).
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