Ⅲ型类人胶原蛋白的重组表达、发酵及其表征
Recombinant expression, fermentation and characterization of a type Ⅲ human like collagen
胶原蛋白是人体中含量最高的蛋白质，具有不可替代的生物学功能，其在食品、美容、医药等领域受到广泛关注且具有极大的市场需求。编码hlCOLⅢα1链的一部分的氨基酸被替换后整合到毕赤酵母基因组中，结合启动子工程和高密度发酵技术，采用两阶段进料方法可使目的蛋白得到可溶性分泌表达，其最高产量为1.05 g L-1。对目的产物进行亲和层析，其纯度可达96%。N/C端蛋白质测序结果与理论值相符，氨基酸分析中Gly占27.02%，Pro占23.92%，符合胶原蛋白的特性。质谱、傅里叶变换红外光谱和圆二色谱分析表明，目标产物在培养基中以同源二聚体或三聚体的形式存在，这也为探索胶原蛋白的自组装研究奠定了基础。
ollagen, the highest content protein in the body, as irreplaceable biological functions, and it is widespread concerned in food, beauty, and medicine with great market demand. The gene encoding hlCOLⅢ α1 chain fragment was integrated into Pichia pastoris genome after partial amino acids were substituted. Combined with promoter engineering and high-density fermentation technology, soluble secretory expression with the highest yield of 1.05 g L-1 was achieved using two-stage feeding method, and the purity could reach 96% after affinity purification. The determination of N/C-terminal protein sequence were consistent with the theoretical expectation, glycine shared 27.02% and proline 23.92% in amino acid analysis, which were in accordance with the characteristics of collagen. Mass spectrometry combined with Fourier transform infrared spectroscopy and circular dichroism revealed that the target product existed as homologous dimer and trimer in the broth , which laid a foundation for exploring the self-assembly of collagen.
史劲松、龚劲松、项志祥、许正宏
生物科学研究方法、生物科学研究技术生物工程学生物化学
类人胶原蛋白分泌表达发酵蛋白纯化表征
human-like collagen secretory expression fermentation protein purification characterization
史劲松,龚劲松,项志祥,许正宏.Ⅲ型类人胶原蛋白的重组表达、发酵及其表征[EB/OL].(2022-05-17)[2025-08-18].http://www.paper.edu.cn/releasepaper/content/202205-93.点此复制
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