文献类型：期刊文献

英文题名：Soluble expression and purification of high-bioactivity recombinant human bone morphogenetic protein-2 by codon optimisation in Escherichia coli

作者：Chen, Wei[1,2];Zhang, Caiqian[1];Wu, Yeqing[2];Su, Xiuping[1]

机构：[1]Shaoxing Univ, Yuanpei Coll, Shaoxing 312000, Zhejiang, Peoples R China;[2]Zhejiang Sci Tech Univ, Coll Life Sci, Hangzhou 310018, Peoples R China

年份：2019

卷号：32

期号：3

起止页码：153

外文期刊名：PROTEIN ENGINEERING DESIGN & SELECTION

收录：SCI-EXPANDED(收录号：WOS:000509500400004)、、WOS

基金：This work was financially supported by the National Science Foundation of Zhejiang Province (LQ16C050002), the Technology Planning Project of Shaoxing (2018C30012) and the Fourth-Batch Innovative Team of Shaoxing (Industrial Textile Innovative Team), Project of Shaoxing University Yuanpei College (KY2018Z01).

语种：英文

外文关键词：bone morphogenetic protein-2; dimer; MG-63 cell; periplasmic space

外文摘要：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.