Expression of codon optimized human bone morphogenetic protein 4 in Pichia pastoris
Expression of codon optimized human bone morphogenetic protein 4 in Pichia pastoris
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密码子优化的人骨形态发生蛋白4在毕赤酵母中的表达
DOI：
10.1002/bab.1146
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发表时间：
2014-03-01
影响因子：
2.8
通讯作者：
Zhang, Yanding
中科院分区：
文献类型：
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作者：
Huang, Yide;Zhen, Binqiong;Zhang, Yanding
骨形态发生蛋白（BMP）是具有治疗潜力的TGF-家族成员蛋白质。天然来源的BMP数量有限，在异源蛋白表达系统中生产具有生物活性的BMP仍然是其临床应用的障碍。本研究根据毕赤酵母中密码子相对同义密码子使用值对人骨形成蛋白4成熟区（hBMP 4）的DNA序列进行了优化，优化后的序列中A+T含量在30%~ 55%之间。在毕赤酵母中，优化序列的hBMP 4蛋白表达量（48 mg/L）比天然序列的表达量（12 mg/L）提高了4倍。重组hBMP 4蛋白经SP Sepharose和肝素亲和层析纯化。通过测量细胞增殖刺激和小鼠模型中异位软骨形成的诱导来检查重组hBMP 4的生物活性。结果表明，优化后的DNA序列能显著提高hBMP 4蛋白在毕赤酵母中的表达量，并产生具有生物学活性的重组hBMP 4，表明优化后的DNA序列具有大规模生产hBMP 4蛋白的潜在临床应用价值。
Bone morphogenetic proteins (BMPs) are TGF- family member proteins that have therapeutic potential. The amount of BMPs from natural resources is limited, and the production of biologically active BMPs in heterologous protein expression systems remains an obstacle for their clinical application. In this study, the DNA sequence of human BMP4 mature domain (hBMP4) was optimized according to the codon relative synonymous codon usage values in Pichia pastoris, and the A+T content in the sequence after optimization was within the range of 30% to 55%. In Pichia pastoris cultured in shake-flask, the expression level of hBMP4 protein from the optimized sequence (48mg/L) increased fourfold in comparison with that from the native sequence (12mg/L). Recombinant hBMP4 protein was purified by SP Sepharose and heparin affinity chromatography. The biological activities of recombinant hBMP4 were examined by measuring proliferation stimulation in cells and induction of ectopic cartilage formation in mouse models. Our results demonstrated that the optimized DNA sequence could significantly enhance hBMP4 protein expression in Pichia pastoris compared with the native sequence and produce biologically active recombinant hBMP4; this indicates the potential of this optimized sequence for bulk production of hBMP4 protein in future clinical applications.