We developed a simple method of preparing recombinant human bone morphogenetic protein-2 (rhBMP-2) with high biological activity. This rhBMP-2 was overproduced in Escherichia coli as a fusion protein with thioredoxin 6xHis-tag at its amino terminus. The cDNA fragment of human bone morphogenetic protein-2 (hBMP-2) fused to the secretion signal of alkaline phosphatase (PhoA) was expressed under T7 promoter in E. coli. After DNA sequence confirmation, the recombinant vector pETpho-bmp2 was transformed into E. coli BL21 (DE3). rhBMP-2 was produced by the recombinant strain pETpho-bmp2/BL21 (DE3) in a soluble form with an yield of 6.2 mg/L culture. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) results showed that the molecular weight of the product was approximately 28 kD. Moreover, rhBMP-2 was secreted as a dimer with a natural structure. rhBMP-2, purified by Ni Nitrilotriacetic acid Agarose (Ni-NTA) affinity chromatography, was used to examine osteosarcoma MG-63 cells and assay the alkaline phosphatase (ALP) activity. Results showed that rhBMP-2 induced MG-63 cell differentiation. When the final concentration was 500 ng/mL, the effect was more remarkable and ALP activity reached 525% compared with that of the control group.

中文翻译：

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高密码子重组人骨形态发生蛋白2的可溶性表达和纯化通过密码子优化在大肠杆菌中。

我们开发了一种简单的制备具有高生物活性的重组人骨形态发生蛋白2（rhBMP-2）的方法。该rhBMP-2在大肠杆菌中过量生产，是在其氨基末端与硫氧还蛋白6xHis-tag的融合蛋白。与碱性磷酸酶（PhoA）分泌信号融合的人骨形态发生蛋白2（hBMP-2）的cDNA片段在大肠杆菌中的T7启动子下表达。DNA序列确认后，将重组载体pETpho-bmp2转化到大肠杆菌BL21（DE3）中。rhBMP-2由重组菌株pETpho-bmp2 / BL21（DE3）产生，可溶形式，产量为6.2 mg / L。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结果表明，该产物的分子量约为28 kD。而且，rhBMP-2分泌为具有天然结构的二聚体。通过镍三乙酸琼脂糖（Ni-NTA）亲和层析纯化的rhBMP-2用于检查骨肉瘤MG-63细胞并测定碱性磷酸酶（ALP）活性。结果显示，rhBMP-2诱导MG-63细胞分化。当最终浓度为500 ng / mL时，与对照组相比，效果更显着，ALP活性达到525％。