人骨形成蛋白2A活性片段在大肠杆菌中的高效表达

将编码人骨形成蛋白２Ａ（ＢＭＰ２Ａ）Ｃ端１７３个氨基酸（ＢＭＰ２３）和１３４个氨基酸（ＢＭＰ２４）的ＤＮＡ基因片段分别重组克隆进入ＰＬ启动子控制下的表达载体，构建了表达质粒ｐＢＬＢＭＰ２３和ｐＢＬＢＭＰ２４，分别转化大肠杆菌进行表达研究．ＳＤＳ－ＰＡＧＥ分析温敏诱导的表达菌，可以分别观察到分子量为２０ｋＤ和１５．５ｋＤ的高表达条带，与理论计算的分子量一致，表达量分别占细菌蛋白质总量的１０％和２０％左右。表达产物经包含体制备达到８０％以上纯度。Ｎ端序列测定的１５个氨基酸，与重组ｃＤＮＡ基因编码的序列相同．ＢＭＰ２３和ＢＭＰ２４包含体经复性处理后，得到二聚体分子蛋白质条带，与骨基质胶原重组后在大鼠体内测活，观察到ＢＭＰ２３诱导软骨细胞生成，ＢＭＰ２４刺激丰富的骨样胶原组织合成．

Expression of C-terminal Segments of Bone Morphogenetic Protein in E.coli

Two recombinant C-terminal segments of human bone morphogenetic protein 2A (BMP2A), designated as BMP23 (178aa) and BMP24 (134aa), were expressed under the control of PL promoter in E. coli. SDW-PAGE analysis showed two induced protein bands at 20kD and 15.5kD, which were about 10% and 20% of the total bacterial protein, respectively.Both the expressed proteins were insoluble aggregates, which reached 80% purity after rapid preparation of the products as the inclusion body. The results of N-terminal amino acid sequencing showed that the first 15 amino acids at the N-terminal were identical to those encoded by the cDNA genes of both BMP23 and BMP24. After refolding botn BMP23 and BMP24formed disulfide-linked dimeric protein bands in SDS-PAGE. Bioassay in vivo revealed that BMP23 induced chondrocytes differentiation and BMP24 stimulated collagen synthesis.